Nanoparticles for monoclonal antibody delivery

ABSTRACT

The invention discloses the nanoparticles composed of chitosan, poly-glutamic acid, and at least one bioactive agent of monoclonal antibody. The nanoparticles are characterized with a positive surface charge and their enhanced permeability for paracellular drug delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 11/398,440, filed Apr. 5, 2006, now U.S. Pat. No.7,381,716, which is a continuation-in-part application of U.S. patentapplication Ser. No. 11/284,734, filed Nov. 21, 2005, now U.S. Pat. No.7,282,194, which is a continuation-in-part application of U.S. patentapplication Ser. No. 11/029,082, filed Jan. 4, 2005, now U.S. Pat. No.7,265,090, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is related to compositions and medical uses ofbiodegradable nanoparticles with monoclonal antibody protein drugs andtheir oral delivery with enhanced paracellular permeability.

BACKGROUND OF THE INVENTION

Production of pharmaceutically active peptides and proteins in largequantities has become feasible (Biomacromolecules 2004; 5:1917-1925).The oral route is considered the most convenient way of drugadministrations for patients. Nevertheless, the intestinal epithelium isa major barrier to the absorption of hydrophilic drugs such as peptidesand proteins (J. Control. Release 1996; 39:131-138). This is becausehydrophilic drugs cannot easily diffuse across the cells through thelipid-bilayer cell membranes. Attentions have been given to improvingparacellular transport of hydrophilic drugs (J. Control. Release 1998;51:35-46). The transport of hydrophilic molecules via the paracellularpathway is, however, severely restricted by the presence of tightjunctions that are located at the luminal aspect of adjacent epithelialcells (Annu. Rev. Nutr. 1995; 15:35-55). These tight junctions form abarrier that limits the paracellular diffusion of hydrophilic molecules.The structure and function of tight junctions is described, inter alia,in Ann. Rev. Physiol. 1998; 60:121-160 and in Ballard T S et al., Annu.Rev. Nutr. 1995; 15:35-55. Tight junctions do not form a rigid barrierbut play an important role in the diffusion through the intestinalepithelium from lumen to bloodstream and vice versa.

Movement of solutes between cells, through the tight junctions that bindcells together into a layer as with the epithelial cells of thegastrointestinal tract, is termed paracellular transport. Paracellulartransport is passive. Paracellular transport depends on electrochemicalgradients generated by transcellular transport and on solvent dragthrough tight junctions. Tight junctions form an intercellular barrierthat separates the apical and basolateral fluid compartments of a celllayer. Movement of a solute through a tight junction from apical tobasolateral compartments depends on the “tightness” of the tightjunction for that solute.

Polymeric nanoparticles have been widely investigated as carriers fordrug delivery (Biomaterials 2002; 23:3193-3201). Much attention has beengiven to the nanoparticles made of synthetic biodegradable polymers suchas poly-ε-caprolactone and polylactide due to their goodbiocompatibility (J. Drug Delivery 2000; 7:215-232; Eur. J. Pharm.Biopharm. 1995; 41:19-25). However, these nanoparticles are not idealcarriers for hydrophilic drugs because of their hydrophobic property.Some aspects of the invention relate to a novel nanoparticle system,composed of hydrophilic chitosan and poly(glutamic acid) hydrogels thatis prepared by a simple ionic-gelation method. This technique ispromising as the nanoparticles are prepared under mild conditionswithout using harmful solvents. It is known that organic solvents maycause degradation of peptide or protein drugs that are unstable andsensitive to their environments (J. Control. Release 2001; 73:279-291).

Following the oral drug delivery route, protein drugs are readilydegraded by the low pH of gastric medium in the stomach. The absorptionof protein drugs following oral administration is challenging due totheir high molecular weight, hydrophilicity, and susceptibility toenzymatic inactivation. Protein drugs at the intestinal epithelium couldnot partition into the hydrophobic membrane and thus can only traversethe epithelial barrier via the paracellular pathway. However, the tightjunction forms a barrier that limits the paracellular diffusion ofhydrophilic molecules.

Chitosan (CS), a cationic polysaccharide, is generally derived fromchitin by alkaline deacetylation (J. Control. Release 2004; 96:285-300).It was reported from literature that CS is non-toxic and soft-tissuecompatible (Biomacromolecules 2004; 5:1917-1925; Biomacromolecules 2004;5:828-833). Additionally, it is known that CS has a special feature ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells (Pharm. Res. 1994; 11:1358-1361).Most commercially available CSs have a quite large molecular weight (MW)and need to be dissolved in an acetic acid solution at a pH value ofapproximately 4.0 or lower that is sometimes impractical. However, thereare potential applications of CS in which a low MW would be essential.Given a low MW, the polycationic characteristic of CS can be usedtogether with a good solubility at a pH value close to physiologicalranges (Eur. J. Pharm. Biopharm. 2004; 57:101-105). Loading of peptideor protein drugs at physiological pH ranges would preserve theirbioactivity. On this basis, a low-MW CS, obtained by depolymerizing acommercially available CS using cellulase, is disclosed herein toprepare nanoparticles of the present invention.

The γ-PGA, an anionic peptide, is a natural compound produced ascapsular substance or as slime by members of the genus Bacillus (Crit.Rev. Biotechnol. 2001; 21:219-232). γ-PGA is unique in that it iscomposed of naturally occurring L-glutamic acid linked together throughamide bonds. It was reported from literature that this naturallyoccurring γ-PGA is a water-soluble, biodegradable, and non-toxicpolymer. A related, but structurally different polymer, [poly(α-glutamicacid), α-PGA] has been used for drug delivery (Adv. Drug Deliver. Rev.2002; 54:695-713; Cancer Res. 1998; 58:2404-2409). α-PGA is usuallysynthesized from poly(γ-benzyl-L-glutamate) by removing the benzylprotecting group with the use of hydrogen bromide. Hashida et al. usedα-PGA as a polymeric backbone and galactose moiety as a ligand to targethepatocytes (J. Control. Release 1999; 62:253-262). Their in vivoresults indicated that the galactosylated α-PGA had a remarkabletargeting ability to hepatocytes and degradation of α-PGA was observedin the liver.

Thanou et al. reported chitosan and its derivatives as intestinalabsorption enhancers (Adv Drug Deliv Rev 2001; 50:S91-S101). Chitosan,when protonated at an acidic pH, is able to increase the paracellularpermeability of peptide drugs across mucosal epithelia.Co-administration of chitosan or trimethyl chitosan chloride withpeptide drugs were found to substantially increase the bioavailabilityof the peptide in animals compared with administrations without thechitosan component.

U.S. Patent Application publication no. 2006/0051424A1, published onMar. 9, 2006, entire contents of which are incorporated herein byreference, discloses nanoparticle compositions comprising a cationicbiopolymer and at least one biologically active substance,pharmaceutical compositions comprising such nanoparticles and methodsfor the oral administration of biologically active molecules which aresusceptible to degradation in the gastro-intestinal tract usingnanoparticle.

U.S. Patent Application publication no. 2005/0042298A1, published onFeb. 24, 2005, entire contents of which are incorporated herein byreference, discloses a functionalized composition for use in forming animmunonanoparticle, the functionalized composition comprising ananoparticle-forming polymer, a polymeric strand having a first endattached to the nanoparticle-forming polymer and a second end, and aconjugation agent attached to the second end of the polymeric strand. Inone embodiment, a functionalized moiety for use in forming theimmunonanoparticle further includes a targeting agent attached to theconjugation agent.

U.S. Pat. No. 6,689,338 B2 issued on Feb. 10, 2004, entire contents ofwhich are incorporated herein by reference, discloses a bioconjugatecomprising a radioactive, metal sulfide or metal oxide nanoparticlecovalently linked to at least one biological vector molecule, whereinthe at least one biological vector molecule is selected from a groupconsisting of monoclonal antibodies (mAb), fragments of monoclonalantibodies, and peptides.

U.S. Pat. No. 6,165,440, issued on Dec. 26, 2000, entire contents ofwhich are incorporated herein by reference, discloses a method ofanti-cancer drug delivery in a solid tumor, comprising the steps ofadministering at least one anti-cancer drug to the tumor, injectingnanoparticles or microparticles to the tumor intravenously, andirradiating the tumor with radiation, wherein the anti-cancer drug isselected from the group consisting of a monoclonal antibody, a cytokine,an antisense oligonucleotide, and a gene-targeting vector.

Currently, most monoclonal antibody is administered to a patient viaintravenous injection or subcutaneous injection. It is desirable toadminister monoclonal antibodies orally in nanoparticles that provideenhanced paracellular permeability, bioavailability, and sustainedrelease, where the nanoparticles biodegrade to biocompatible byproductsin situ.

SUMMARY OF THE INVENTION

It is one object of the present invention to provide a novelnanoparticle system and methods of preparation for paracellulartransport drug delivery using a simple and mild ionic-gelation methodupon addition of a poly-γ-glutamic acid (γ-PGA) solution into a lowmolecular weight chitosan (low-MW CS) solution. In one embodiment, themolecular weight of a low-MW CS of the present invention is about 80 kDaor less, preferably at about 40 kDa, adapted for adequate solubility ata pH that maintains the bioactivity of protein and peptide drugs. It isstipulated that a chitosan particle with about 30-50 kDa molecularweight is kidney inert. The particle size and the zeta potential valueof the prepared nanoparticles are controlled by their constitutedcompositions.

The results obtained by the TEM (transmission electron microscopy) andAFM (atomic force microscopy) examinations showed that the morphology ofthe prepared nanoparticles was generally spherical in shape. Evaluationof the prepared nanoparticles in enhancing intestinal paracellulartransport was investigated in vitro in Caco-2 cell monolayers. In someaspects of the present invention, it provides the nanoparticles with CSdominated on the surfaces to effectively reduce the transepithelialelectrical resistance (TEER) of Caco-2 cell monolayers. The confocallaser scanning microscopy (CLSM) observations confirm that thenanoparticles with CS dominated on the surface are able to open thetight junctions between Caco-2 cells and allows transport of thenanoparticles via the paracellular pathways.

In some application, a normal or high molecular weight chitosan is usedin preparing the nanoparticles.

Some aspects of the invention relate to a method of enhancing intestinalor blood brain paracellular transport configured for delivering at leastone bioactive agent in a patient comprising administering nanoparticlescomposed of γ-PGA and chitosan, wherein the step of administering thenanoparticles may be via oral administration. In one embodiment, thechitosan dominates on a surface of the nanoparticles as shell substrateand the negatively charged γ-PGA as core substrate. In anotherembodiment, a substantial surface of the nanoparticles is characterizedwith a positive surface charge. In a further embodiment, thenanoparticles of the present invention comprise at least one positivelycharged shell substrate and at least one negatively charged coresubstrate. In one embodiment, at least one bioactive or protein drug isconjugated with the negatively charged core substrate.

In a further embodiment, the chitosan of the nanoparticles is a lowmolecular weight chitosan, wherein the low molecular weight chitosan hasa molecular weight of about 50 kDa, preferably having a molecular weightof less than about 40 kDa.

In a further embodiment, the nanoparticles have a mean particle sizebetween about 50 and 400 nanometers, preferably between about 100 and300 nanometers, and most preferably between about 100 and 200nanometers.

In some embodiments, the nanoparticles are loaded with a therapeuticallyeffective amount of at least one bioactive agent, wherein the bioactiveagent is selected from a group consisting of proteins (for example,monoclonal antibodies), peptides, nucleosides, nucleotides, antiviralagents, antineoplastic agents, antibiotics, and anti-inflammatory drugs.Further, the bioactive agent may be selected from a group consisting ofcalcitonin, cyclosporin, insulin, oxytocin, tyrosine, enkephalin,tyrotropin releasing hormone, follicle stimulating hormone, luteinizinghormone, vasopressin and vasopressin analogs, catalase, superoxidedismutase, interleukin-II, interferon, colony stimulating factor, tumornecrosis factor (TNF) and melanocyte-stimulating hormone. In onepreferred embodiment, the bioactive agent is an Alzheimer antagonist.

Some aspects of the invention relate to an oral dose of nanoparticlesthat effectively enhance intestinal or blood brain paracellulartransport comprising γ-PGA or α-PGA and low molecular weight chitosan,wherein the chitosan dominates on a surface of the nanoparticles. Someaspects of the invention relate to an oral dose of nanoparticles thateffectively enhance intestinal or blood brain paracellular transportcomprising a negative component, such as γ-PGA, α-PGA, heparin, orheparan sulfate, in the core and low molecular weight chitosan, whereinthe chitosan dominates on a surface of the nanoparticles with positivecharges. In a further embodiment, the nanoparticles comprise at leastone bioactive agent, such as insulin, insulin analog, Alzheimer'sdisease antagonist, Parkison's disease antagonist, or otherprotein/peptide. The bioactive agent for treating Alzheimer's diseasemay include memantine hydrochloride (Axura® by Merz Pharmaceuticals),donepezil hydrochloride (Aricept® by Eisai Co. Ltd.), rivastigminetartrate (Exelon® by Novartis), galantamine hydrochloride (Reminyl® byJohnson & Johnson), and tacrine hydrochloride (Cognex® by Parke Davis).Examples of insulin or insulin analog products include, but not limitedto, Humulin® (by Eli Lilly), Humalog® (by Eli Lilly) and Lantus® (byAventis).

Some aspects of the invention relate to an oral dose of nanoparticlesthat effectively enhance intestinal or blood brain paracellulartransport comprising γ-PGA and low molecular weight chitosan, whereinthe nanoparticles are crosslinked with a crosslinking agent or withlight, such as ultraviolet irradiation.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle. In oneembodiment, the third component is γ-PGA, α-PGA, derivatives or salts ofPGA, heparin, glycosaminoglycans or alginate. In another embodiment, thefirst component comprises insulin at a concentration range of 0.075 to0.091 mg/ml, the second component at a concentration range of 0.67 to0.83 mg/ml, and the third component comprises γ-PGA at a concentrationrange of 0.150 to 0.184 mg/ml.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein theat least one bioactive agent is an antagonist for Alzheimer's disease oris for treating Alzheimer's disease selected from the group consistingof memantine hydrochloride, donepezil hydrochloride, rivastigminetartrate, galantamine hydrochloride, and tacrine hydrochloride. In afurther embodiment, the at least one bioactive agent is insulin orinsulin analog. In still another embodiment, the at least one bioactiveagent is selected from the group consisting of proteins, peptides,nucleosides, nucleotides, antiviral agents, antineoplastic agents,antibiotics, and anti-inflammatory drugs.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, wherein the nanoparticles are further encapsulated in agelcap.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein thesecond component is crosslinked. In one embodiment, the degree ofcrosslinking is less than 50%. In another embodiment, the degree ofcrosslinking is ranged between 1% and 20%.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, each nanoparticle comprising a first component of at leastone bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle, wherein thesecond component is crosslinked with a crosslinking agent selected fromthe group consisting of genipin, its derivatives, analog, stereoisomersand mixtures thereof. In one embodiment, the crosslinking agent isselected from the group consisting of epoxy compounds, dialdehydestarch, glutaraldehyde, formaldehyde, dimethyl suberimidate,carbodiimides, succinimidyls, diisocyanates, acyl azide, reuterin,ultraviolet irradiation, dehydrothermal treatment,tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine andphoto-oxidizers.

Some aspects of the invention provide a dose of nanoparticlescharacterized by enhancing intestinal or brain blood paracellulartransport, wherein the low molecule weight chitosan has a molecularweight of 80 kDa or less. In one embodiment, the low molecule weightchitosan is further grafted with a polymer having a chemical formula as:

where R is ≧12

Some aspects of the invention provide a method of enhancing intestinalor brain blood paracellular transport comprising administering a dose ofnanoparticles, wherein each nanoparticle comprises a first component ofat least one bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein thesecond component dominates on a surface of the nanoparticle. In oneembodiment, the step of administering the dose of nanoparticles is viaoral administration for enhancing intestinal paracellular transport. Inanother embodiment, the step of administering the dose of nanoparticlesis via venous administration or injection for enhancing brain bloodparacellular transport or reducing the blood-brain barrier (BBB).

Some aspects of the invention provide a method of treating diabetes of apatient comprising orally administering insulin containing nanoparticleswith a dosage effective amount of the insulin to treat the diabetes,wherein at least a portion of the nanoparticles comprises a positivelycharged shell substrate and a negatively charged core substrate. In oneembodiment, the shell substrate comprises chitosan, chitin, chitosanoligosaccharides, and chitosan derivatives thereof, wherein asubstantial portion of a surface of the nanoparticles is characterizedwith a positive surface charge. In another embodiment, the coresubstrate is selected from a group consisting of γ-PGA, α-PGA,water-soluble salts of PGA, metal salts of PGA, heparin, heparinanalogs, low molecular weight heparin, glycosaminoglycans, and alginate.The molecular formula of the insulin is selected from a group consistingof C₂₅₄H₃₇₇N₆₅O₇₅S₆, C₂₅₇H₃₈₃N₆₅O₇₇S₆, C₂₅₆H₃₈₁N₆₅O₇₉S₆,C₂₆₇H₄₀₄N₇₂O₇₈S₆, and the like.

In one embodiment, the orally administering insulin containingnanoparticles comprise a dosage effective amount of the insulin to treatthe diabetes comprising an insulin amount of between about 15 units to45 units, preferably between about 25 units to 35 units, per kilogrambody weight of the patient. In a further embodiment, theinsulin-containing nanoparticle comprises a trace amount of zinc orcalcium, or is treated with enteric coating.

In one embodiment, the insulin containing nanoparticles further compriseat least one paracellular transport enhancer, wherein the paracellulartransport enhancer may be selected from a group consisting of Ca²⁺chelators, bile salts, anionic surfactants, medium-chain fatty acids,and phosphate esters. In another embodiment, the nanoparticles and theparacellular transport enhancer are co-encapsulated in a softgel capsuleor are encapsulated separately.

Some aspects of the invention provide nanoparticles for oraladministration in a patient, comprising a positively charged shellsubstrate, a negatively charged core substrate, and a bioactive agentconjugated with the core substrate, wherein the core substrate isselected from a group consisting of heparin, heparin analogs, lowmolecular weight heparin, glycosaminoglycans, and alginate, thebioactive agent being selected from a group consisting of chondroitinsulfate, hyaluronic acid, growth factor and protein withpharmaceutically effective amount.

Some aspects of the invention provide nanoparticles for oraladministration in a patient, comprising a positively charged shellsubstrate, a negatively charged core substrate, and a bioactive agentconjugated with the core substrate, wherein the bioactive agent iscalcitonin or vancomycin.

Some aspects of the invention provide a method of treating Alzheimer'sdiseases of a patient comprising intravenously administering bioactivenanoparticles with a dosage effective to treat the Alzheimer's diseases,wherein the bioactive nanoparticles comprises a positively charged shellsubstrate, a negatively charged core substrate, and at least onebioactive agent for treating Alzheimer's disease, wherein the at leastone bioactive agent is selected from a group consisting of memantinehydrochloride, donepezil hydrochloride, rivastigmine tartrate,galantamine hydrochloride, and tacrine hydrochloride.

In one embodiment, the dosage effective to treat the Alzheimer'sdiseases comprises administering the at least one bioactive agent fortreating Alzheimer's disease at about 10 mg to 40 mg per day over aperiod of one month to one year. In another embodiment, at least aportion the shell substrate is crosslinked, preferably at a degree ofcrosslinking less than about 50%, or most preferably between about 1%and 20%.

Some aspects of the invention provide a method of treating a targettissue or organ of a patient with a monoclonal antibody, comprising thesteps of: providing the monoclonal antibody to the tissue or organ,wherein the monoclonal antibody is encapsulated within nanoparticles;administering the nanoparticles to the patient orally; and treating thetarget tissue or organ with the monoclonal antibody that is sustainedreleased from the nanoparticles. In one embodiment, the monoclonalantibody is an anti-cancer drug. In another embodiment, the monoclonalantibody is Adalimumab or Bevacizumab.

Some aspects of the invention provide nanoparticles for oraladministration in a patient, comprising a positively charged shellsubstrate, a negatively charged core substrate, and a monoclonalantibody encapsulated within the shell substrate. In one preferredembodiment, the monoclonal antibody is mixed with, conjugated or coupledto, the core substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional objects and features of the present invention will becomemore apparent and the disclosure itself will be best understood from thefollowing Detailed Description of the Exemplary Embodiments, when readwith reference to the accompanying drawings.

FIG. 1 shows GPC chromatograms of (a) standard-MW CS beforedepolymerization and the low-MW CS after depolymerization; (b) thepurified γ-PGA obtained from microbial fermentation.

FIG. 2 shows (a) FT-IR and (b) ¹H-NMR spectra of the purified γ-PGAobtained from microbial fermentation.

FIG. 3 shows FT-IR spectra of the low-MW CS and the prepared CS-γ-PGAnanoparticles.

FIG. 4 shows (a) a TEM micrograph of the prepared CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) an AFM micrograph of the preparedCS-γ-PGA nanoparticles (0.01% γ-PGA:0.01% CS).

FIG. 5 shows changes in particle size and zeta potential of (a) theCS-γ-PGA nanoparticles (0.10% γ-PGA:0.20% CS) and (b) the CS-γ-PGAnanoparticles (0.10% γ-PGA:0.01% CS) during storage for up to 6 weeks.

FIG. 6 shows effects of the prepared CS-γ-PGA nanoparticles on the TEERvalues of Caco-2 cell monolayers.

FIG. 7 shows fluorescence images (taken by an inversed confocal laserscanning microscope) of 4 optical sections of a Caco-2 cell monolayerthat had been incubated with the fCS-γ-PGA nanoparticles with a positivesurface charge (0.10% γ-PGA:0.20% CS) for (a) 20 min and (b) 60 min.

FIG. 8 shows an illustrative protein transport mechanism through a celllayer, including transcellular transport and paracelluler transport.

FIG. 9 shows a schematic illustration of a paracellular transportmechanism.

FIG. 10 shows an fCS-γ-PGA nanoparticle with FITC-labeled chitosanshaving positive surface charge.

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA.

FIG. 12 shows loading capacity and association efficiency of insulin innanoparticles of chitosan as reference.

FIG. 13 shows the stability of insulin-loaded nanoparticles.

FIG. 14 shows a representative in vitro study with insulin drug releaseprofile in a pH-adjusted solution.

FIG. 15 shows the bioavailability of insulin of orally administeredinsulin-loaded nanoparticles in diabetic rats.

FIG. 16 shows a proposed mechanism of nanoparticles released from theenteric coating.

FIG. 17 shows the schematic illustration of insulin conjugated withhistidine and/or glutamic acid side groups of the γ-PGA via zinc.

FIG. 18 shows the schematic illustration of insulin conjugated with acarboxyl side group of the γ-PGA via zinc.

FIG. 19 shows a schematic composition of a nanoparticle with a shellsubstrate and a core substrate having a monoclonal antibody.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The preferred embodiments of the present invention described belowrelate particularly to preparation of nanoparticles composed ofchitosan/poly-glutamic acid/insulin and their permeability to enhancethe intestinal or blood brain paracellular permeation by opening thetight junctions between epithelial cells. While the description setsforth various embodiment specific details, it will be appreciated thatthe description is illustrative only and should not be construed in anyway as limiting the invention. Furthermore, various applications of theinvention, and modifications thereto, which may occur to those who areskilled in the art, are also encompassed by the general conceptsdescribed below.

γ-PGA is a naturally occurring anionic homo-polyamide that is made ofL-glutamic acid units connected by amide linkages between α-amino andγ-carboxylic acid groups (Crit. Rev. Biotechnol. 2001; 21:219-232). Itis an exocellular polymer of certain Bacillus species that is producedwithin cells via the TCA cycle and is freely excreted into thefermentation broth. Its exact biological role is not fully known,although it is likely that γ-PGA is linked to increasing the survival ofproducing strains when exposed to environmental stresses. Because of itswater-solubility, biodegradability, edibility, and non-toxicity towardhumans and the environment, several applications of γ-PGA in food,cosmetics, medicine, and water treatment have been investigated in thepast few years.

EXAMPLE NO. 1

Materials and Methods of Nanoparticles Preparation

CS (MW˜2.8×10⁵) with a degree of deacetylation of approximately 85% wasacquired from Challenge Bioproducts Co. (Taichung, Taiwan). Acetic acid,cellulase (1.92 units/mg), fluorescein isothiocyanate (FITC), phosphatebuffered saline (PBS), periodic acid, sodium acetate, formaldehyde,bismuth subnitrate, and Hanks balanced salt solution (HBSS) werepurchased from Sigma Chemical Co. (St. Louis, Mo.). Ethanol absoluteanhydrous and potassium sodium tartrate were obtained from Merck(Darmstadt, Germany). Non-essential amino acid (NEAA) solution, fetalbovine serum (FBS), gentamicin and trypsin-EDTA were acquired from Gibco(Grand Island, N.Y.). Eagle's minimal essential medium (MEM) waspurchased from Bio West (Nuaille, France). All other chemicals andreagents used were of analytical grade.

EXAMPLE NO. 2

Depolymerization of CS by Enzymatic Hydrolysis

Regular CS was treated with enzyme (cellulase) to produce low-MW CSaccording to a method described by Qin et al. with some modifications(Food Chem. 2004; 84:107-115). A solution of CS (20 g/l) was prepared bydissolving CS in 2% acetic acid. Care was taken to ensure totalsolubility of CS. Then, the CS solution was introduced into a vessel andadjusted to the desired pH 5.0 with 2N aqueous NaOH. Subsequently,cellulase (0.1 g) was added into the CS solution (100 ml) andcontinuously stirred at 37° C. for 12 hours. Afterward, thedepolymerized CS was precipitated with aqueous NaOH at pH 7.0-7.2 andthe precipitated CS washed three times with deionized water. Theresulting low-MW CS was lyophilized in a freeze dryer (Eyela Co. Ltd,Tokyo, Japan).

The average molecular weight of the depolymerized CS was determined by agel permeation chromatography (GPC) system equipped with a series of PLaquagel-OH columns (one Guard 8 μm, 50×7.5 mm and two MIXED 8 μm,300×7.5 mm, PL Laboratories, UK) and a refractive index (RI) detector(RI2000-F, SFD, Torrance, Calif.). Polysaccharide standards (molecularweights range from 180 to 788,000, Polymer Laboratories, UK) were usedto construct a calibration curve. The mobile phase contained 0.01MNaH₂PO₄ and 0.5M NaNO₃ and was brought to a pH of 2.0. The flow rate ofmobile phase was 1.0 ml/min, and the columns and the RI detector cellwere maintained at 30° C.

Factors limiting applications of most commercially available CSs aretheir high molecular weight and thus high viscosity and poor solubilityat physiological pH ranges. Low-MW CS overcomes these limitations andhence finds much wider applications in diversified fields. It wassuggested that low-MW CS be used as a parenteral drug carrier due to itslower antigen effect (Eur. J. Pharm. Biopharm. 2004; 57:101-105). Low-MWCS was used as a non-viral gene delivery system and showed promisingresults (Int. J. Pharm. 1999; 178:231-243). Other studies based onanimal testing showed the possibilities of low-MW CS for treatment oftype 2 diabetes and gastric ulcer (Biol. Pharm. Bull. 2002; 25:188-192).Several hydrolytic enzymes such as lysozyme, pectinase, cellulase,bromelain, hemicellulase, lipase, papain and the like can be used todepolymerize CS (Biochim. Biophys. Acta 1996; 1291:5-15; Biochem. Eng.J. 2001; 7:85-88; Carbohydr. Res. 1992; 237:325-332). FIG. 1 a shows GPCchromatograms of both standard-MW (also known as regular-MW) and low-MWCS. It is known that cellulase catalyzes the cleavage of the glycosidiclinkage in CS (Food Chem. 2004; 84:107-115). The low-MW CS used in thestudy was obtained by precipitating the depolymerized CS solution withaqueous NaOH at pH 7.0-7.2. Thus obtained low-MW CS had a MW of about 50kDa (FIG. 1 a). In a preferred embodiment, the low molecular weightchitosan has a molecular weight of less than about 40 kDa, but above 10kDa. Other forms of chitosan may also be applicable, including chitin,chitosan oligosaccharides, and derivatives thereof.

It was observed that the obtained low-MW CS can be readily dissolved inan aqueous solution at pH 6.0, while that before depolymerization needsto be dissolved in an acetic acid solution with a pH value about 4.0.Additionally, it was found that with the low-MW CS, the preparednanoparticles had a significantly smaller size with a narrowerdistribution than their counterparts prepared with the high-MW (alsoknown as standard-MW) CS (before depolymerization), due to its lowerviscosity. As an example, upon adding a 0.10% γ-PGA aqueous solutioninto a 0.20% high-MW CS solution (viscosity 5.73±0.08 cp, measured by aviscometer), the mean particle size of the prepared nanoparticles was878.3±28.4 nm with a polydispersity index of 1.0, whereas adding a 0.10%γ-PGA aqueous solution into the low-MW CS solution (viscosity 1.29±0.02cp) formed nanoparticles with a mean particle size of 218.1±4.1 nm witha polydispersity index of 0.3 (n=5).

EXAMPLE NO. 3

Production and Purification of γ-PGA

γ-PGA was produced by Bacillus licheniformis (ATCC 9945, BioresourcesCollection and Research Center, Hsinchu, Taiwan) as per a methodreported by Yoon et al. with slight modifications (Biotechnol. Lett.2000; 22:585-588). Highly mucoid colonies (ATCC 9945a) were selectedfrom Bacillus licheniformis (ATCC 9945) cultured on the E medium(ingredients comprising L-glutamic acid, 20.0 g/l; citric acid, 12.0g/l; glycerol, 80.0 g/l; NH₄Cl, 7.0 g/l; K₂HPO₄, 0.5 μl; MgSO₄.7H₂O, 0.5g/l; FeCl₃.6H₂O, 0.04 g/l; CaCl₂.2H₂O, 0.15 g/l; MnSO₄.H₂O, 0.104 g/l,pH 6.5) agar plates at 37° C. for several times. Subsequently, youngmucoid colonies were transferred into 10 ml E medium and grown at 37° C.in a shaking incubator at 250 rpm for 24 hours. Afterward, 500 μl ofculture broth was mixed with 50 ml E medium and was transferred into a2.5-1 jar-fermentor (KMJ-2B, Mituwa Co., Osaka, Japan) containing 950 mlof E medium. Cells were cultured at 37° C. The pH was controlled at 6.5by automatic feeding of 25% (v/v) NH₄OH and/or 2M HCl. The dissolvedoxygen concentration was initially controlled at 40% of air saturationby supplying air and by controlling the agitation speed up to 1000 rpm.

After 40 hours, cells were separated from the culture broth bycentrifugation for 20 minutes at 12,000×g at 4° C. The supernatantcontaining γ-PGA was poured into 4 volumes of methanol and leftovernight with gentle stirring. The resulting precipitate containingcrude γ-PGA was collected by centrifugation for 40 minutes at 12,000×gat 4° C. and then was dissolved in deionized water to remove insolubleimpurities by centrifugation for 20 minutes at 24,000×g at 4° C. Theaqueous γ-PGA solution was desalted by dialysis (MWCO: 100,000, SpectrumLaboratories, Inc., Laguna Hills, Calif.) against distilled water for 12hours with water exchanges several times, and finally was lyophilized toobtain pure γ-PGA.

The purified γ-PGA was verified by the proton nuclear magnetic resonance(¹H-NMR) and the FT-IR analyses. Analysis of ¹H-NMR was conducted on anNMR spectrometer (Varian Unityionva 500 NMR Spectrometer, Mo.) usingDMSO-d₆ at 2.49 ppm as an internal reference. Test samples used for theFT-IR analysis first were dried and ground into a powder form. Thepowder then was mixed with KBr (1:100) and pressed into a disk. Analysiswas performed on an FT-IR spectrometer (Perkin Elmer Spectrum RX1 FT-IRSystem, Buckinghamshire, England). The samples were scanned from400-4000 cm⁻¹. The average molecular weight of the purified γ-PGA wasdetermined by the same GPC system as described before. Polyethyleneglycol (molecular weights of 106-22,000) and polyethylene oxide(molecular weights of 20,000-1,000,000, PL Laboratories) standards wereused to construct a calibration curve. The mobile phase contained 0.01MNaH₂PO₄ and 0.2M NaNO₃ and was brought to a pH of 7.0.

The purified γ-PGA obtained from fermentation was analyzed by GPC,¹H-NMR, and FT-IR. As analyzed by GPC (FIG. 1 b), the purified γ-PGA hada MW of about 160 kDa. In the FT-IR spectrum (FIG. 2 a), acharacteristic peak at 1615 cm⁻¹ for the associated carboxylic acid salt(—COO⁻ antisymmetric stretch) on γ-PGA was observed. The characteristicabsorption due to C═O in secondary amides (amide I band) was overlappedby the characteristic peak of —COO⁻. Additionally, the characteristicpeak observed at 3400 cm⁻¹ was the N—H stretch of γ-PGA. In the ¹H-NMRspectrum (FIG. 2 b), six chief signals were observed at 1.73 and 1.94ppm (β-CH₂), 2.19 ppm (γ-CH₂), 4.14 ppm (α-CH), 8.15 ppm (amide), and12.58 ppm (COOH). These results indicated that the observed FT-IR and¹H-NMR spectra correspond well to those expected for γ-PGA.Additionally, the fermented product after purification showed nodetected macromolecular impurities by the ¹H-NMR analysis, suggestingthat the obtained white power of γ-PGA is highly pure.

EXAMPLE NO. 4

Preparation of the CS-γ-PGA Nanoparticles

Nanoparticles were obtained upon addition of γ-PGA aqueous solution (pH7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTechScientific Inc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10ml) at varying concentrations (0.01%, 0.05%, 0.10%, 0.15%, or 0.20% byw/v) under magnetic stirring at room temperature. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Supernatantswere discarded and nanoparticles were resuspended in deionized water forfurther studies. FT-IR was used to analyze peak variations of aminogroups of low-MW CS and carboxylic acid salts of γ-PGA in the CS-γ-PGAnanoparticles.

As stated, nanoparticles were obtained instantaneously upon addition ofa γ-PGA aqueous solution (pH 7.4) into a low-MW CS aqueous solution (pH6.0) under magnetic stirring at room temperature. FIG. 3 shows the FT-IRspectra of the low-MW CS and the CS-γ-PGA nanoparticles. As shown in thespectrum of CS, the characteristic peak observed at 1563 cm⁻¹ was theprotonated amino group (—NH₃ ⁺ deformation) on CS. In the spectrum ofCS-γ-PGA complex, the characteristic peak at 1615 cm⁻¹ for —COO⁻ onγ-PGA disappeared and a new peak at 1586 cm⁻¹ appeared, while thecharacteristic peak of —NH₃ ⁺ deformation on CS at 1563 cm⁻¹ shifted to1555 cm⁻¹. These observations are attributed to the electrostaticinteraction between the negatively charged carboxylic acid salts (—COO⁻)on γ-PGA and the positively charged amino groups (—NH₃ ⁺) on CS (Int. J.Pharm. 2003; 250:215-226). The electrostatic interaction between the twopolyelectrolytes (γ-PGA and CS) instantaneously induced the formation oflong hydrophobic segments (or at least segments with a high density ofneutral ion-pairs), and thus resulted in highly neutralized complexesthat segregated into colloidal nanoparticles (Langmuir. 2004;20:7766-7778).

EXAMPLE NO. 5

Characterization of the CS-γ-PGA Nanoparticles

The morphological examination of the CS-γ-PGA nanoparticles wasperformed by TEM (transmission electron microscopy) and AFM (atomicforce microscopy). The TEM sample was prepared by placing a drop of thenanoparticle solution onto a 400 mesh copper grid coated with carbon.About 2 minutes after deposition, the grid was tapped with a filterpaper to remove surface water and positively stained by using analkaline bismuth solution (Microbiol. Immunol. 1986; 30:1207-1211). TheAFM sample was prepared by casting a drop of the nanoparticle solutionon a slide glass and then dried in vacuum. The size distribution andzeta potential of the prepared nanoparticles were measured using aZetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK).

During storage, aggregation of nanoparticles may occur and thus leads tolosing their structural integrity or forming precipitation ofnanoparticles (Eur. J. Pharm. Sci. 1999; 8:99-107). Therefore, thestability of nanoparticles during storage must be evaluated. In thestability study, the prepared nanoparticles suspended in deionized water(1 mg/ml) were stored at 4° C. and their particle sizes and zetapotential values were monitored by the same Zetasizer as mentionedearlier during storage.

In the preparation of nanoparticles, samples were visually analyzed andthree distinct solution systems were identified: clear solution,opalescent suspension, and solution with precipitation of aggregates.Examined by the Zetasizer, nanoparticles were found in the clearsolution and the opalescent suspension rather than in the solution withprecipitation of aggregates.

The particle sizes and the zeta potential values of CS-γ-PGAnanoparticles, prepared at varying concentrations of γ-PGA and CS, weredetermined and the results are shown in Tables 1a and 1b. It was foundthat the particle size and the zeta potential value of the preparednanoparticles were mainly determined by the relative amount of the localconcentration of γ-PGA in the added solution to the surroundingconcentration of CS in the sink solution. At a fixed concentration ofCS, an increase in the γ-PGA concentration allowed γ-PGA moleculesinteracting with more CS molecules, and thus formed a lager size ofnanoparticles (Table 1a, p<0.05). When the amount of CS moleculesexceeded that of local γ-PGA molecules, some of the excessive CSmolecules were entangled onto the surfaces of CS-γ-PGA nanoparticles.

Thus, the resulting nanoparticles may display a structure of a neutralpolyelectrolyte-complex core surrounded by a positively charged CS shell(Table 1b) ensuring the colloidal stabilization (Langmuir. 2004;20:7766-7778). In contrast, as the amount of local γ-PGA moleculessufficiently exceeded that of surrounding CS molecules, the formednanoparticles had γ-PGA exposed on the surfaces and thus had a negativecharge of zeta potential. Therefore, the particle size and the zetapotential value of the prepared CS-γ-PGA nanoparticles can be controlledby their constituted compositions. The results obtained by the TEM andAFM examinations showed that the morphology of the preparednanoparticles was spherical in shape with a smooth surface (FIGS. 4 aand 4 b). Some aspects of the invention relate to nanoparticles having amean particle size between about 50 and 400 nanometers, preferablybetween about 100 and 300 nanometers, and most preferably between about100 and 200 nanometers. The morphology of the nanoparticles showsspherical in shape with a smooth surface at any pH between 2.5 and 6.6.In one embodiment, the stability of the nanoparticles of the presentinvention at a low pH around 2.5 enables the nanoparticles to be intactwhen exposed to the acidic medium in the stomach.

TABLE 1a Effects of concentrations of γ-PGA and CS on the particle sizesof the prepared CS-γ-PGA nanoparticles Mean Particle Size (nm, n = 5) CSγ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%^(b))  79.0 ± 3.0 103.1 ±4.6  96.7 ± 1.9 103.6 ± 1.9 140.5 ± 2.0 0.05% 157.4 ± 1.7 120.8 ± 3.9144.5 ± 2.4 106.2 ± 3.8 165.4 ± 1.7 0.10% 202.2 ± 3.1 232.6 ± 1.2 161.0± 1.8 143.7 ± 2.7 218.1 ± 4.1 0.15% 277.7 ± 3.2 264.9 ± 2.1 188.6 ± 2.9178.0 ± 2.2 301.1 ± 6.4 0.20% 284.1 ± 2.1 402.2 ± 4.0 ▴ 225.5 ± 3.1365.5 ± 5.1 ^(a))concentration of CS (by w/v) ^(b))concentration ofγ-PGA (by w/v) ▴ precipitation of aggregates was observed

TABLE 1b Effects of concentrations of γ-PGA and CS on the zeta potentialvalues of the prepared CS-γ-PGA nanoparticles. Zeta Potential (mV, n =5) CS γ-PGA 0.01%^(a)) 0.05% 0.10% 0.15% 0.20% 0.01%   15.4 ± 0.3 22.8 ±0.5 19.8 ± 1.5 16.5 ± 1.4 17.2 ± 1.6 0.05% −32.7 ± 0.7 23.7 ± 1.7 27.6 ±0.7 20.3 ± 0.8 19.2 ± 0.6 0.10% −33.1 ± 1.3 21.1 ± 1.6 20.3 ± 1.1 23.6 ±0.9 24.7 ± 1.2 0.15% −33.2 ± 2.1 −21.9 ± 2.0   19.2 ± 0.4 16.9 ± 1.719.8 ± 0.3 0.20% −34.5 ± 0.5 −34.6 ± 0.3   ▴ 14.6 ± 0.7 16.3 ± 0.7^(a))concentration of CS (by w/v) ^(b))concentration of γ-PGA (by w/v) ▴precipitation of aggregates was observed

Two representative groups of the prepared nanoparticles were selectedfor the stability study: one with a positive surface charge (0.10%γ-PGA:0.20% CS) and the other with a negative surface charge (0.10%γ-PGA:0.01% CS). FIG. 5 shows changes in particle size (▪, meandiameter) and zeta potential (●) of (a) the CS-γ-PGA nanoparticles(0.10% γ-PGA:0.20% CS) and (b) the CS-γ-PGA nanoparticles (0.10%γ-PGA:0.01% CS) during storage for up to 6 weeks. It was found thatneither aggregation nor precipitation of nanoparticles was observedduring storage for up to 6 weeks, as a result of the electrostaticrepulsion between the positively charged CS-γ-PGA nanoparticles (for theformer group) or the negatively charged CS-γ-PGA nanoparticles (for thelatter group).

Additionally, changes in particle size and zeta potential of thenanoparticles were minimal for both studied groups (FIGS. 5 a and 5 b).These results demonstrated that the prepared nanoparticles suspended indeionized water were stable during storage.

EXAMPLE NO. 6

Caco-2 Cell Cultures and TEER Measurements

Caco-2 cells were seeded on the tissue-culture-treated polycarbonatefilters (diameter 24.5 mm, growth area 4.7 cm²) in Costar Transwell 6wells/plates (Corning Costar Corp., NY) at a seeding density of 3×10⁵cells/insert. MEM (pH 7.4) supplemented with 20% FBS, 1% NEAA, and 40μg/ml antibiotic-gentamicin was used as the culture medium, and added toboth the donor and acceptor compartments. The medium was replaced every48 hours for the first 6 days and every 24 hours thereafter. Thecultures were kept in an atmosphere of 95% air and 5% CO₂ at 37° C. andwere used for the paracellular transport experiments 18-21 days afterseeding (TEER values in the range of 600-800 Ωcm²).

TEER values of the Caco-2 cell monolayers were monitored with aMillicell®-Electrical Resistance System (Millipore Corp., Bedford,Mass.) connected to a pair of chopstick electrodes. To initiate thetransport experiments, the culture media in the donor and acceptorcompartments were aspirated, and the cells were rinsed twice withpre-warmed transport media (HBSS supplemented with 25 mM glucose, pH6.0). Following a 30-min equilibration with the transport media at 37°C., the cells were incubated for 2 hours with 2 ml transport mediacontaining 0.5 ml test nanoparticle solutions (0.2 mg/ml) at 37° C.Subsequently, solutions of nanoparticles were carefully removed andcells were washed three times with HBSS and replaced by fresh culturemedia. The TEER was measured for another 20 hours to study reversibilityof the effect of test nanoparticles on Caco-2 cell monolayers (Eur. J.Pharm. Sci. 2000; 10:205-214).

The intercellular tight junction is one of the major barriers to theparacellular transport of macromolecules (J. Control. Release 1996;39:131-138; J. Control. Release 1998; 51:35-46). Trans-epithelial iontransport is contemplated to be a good indication of the tightness ofthe junctions between cells and was evaluated by measuring TEER ofCaco-2 cell monolayers in the study. It was reported that themeasurement of TEER can be used to predict the paracellular transport ofhydrophilic molecules (Eur. J. Pharm. Biopharm. 2004; 58:225-235). Whenthe tight junctions open, the TEER value will be reduced due to thewater and ion passage through the paracellular route. Caco-2 cellmonolayers have been widely used as an in vitro model to evaluate theintestinal paracellular permeability of macromolecules.

Effects of the prepared CS-γ-PGA nanoparticles on the TEER values ofCaco-2 cell monolayers are shown in FIG. 6. As shown, the preparednanoparticles with a positive surface charge (CS dominated on thesurface, 0.01% γ-PGA:0.05% CS, 0.10% γ-PGA:0.2% CS, and 0.20%γ-PGA:0.20% CS) were able to reduce the values of TEER of Caco-2 cellmonolayers significantly (p<0.05). After a 2-hour incubation with thesenanoparticles, the TEER values of Caco-2 cell monolayers were reduced toabout 50% of their initial values as compared to the control group(without addition of nanoparticles in the transport media). Thisindicated that the nanoparticles with CS dominated on the surfaces couldeffectively open the tight junctions between Caco-2 cells, resulting ina decrease in the TEER values. It was reported that interaction of thepositively charged amino groups of CS with the negatively charged siteson cell surfaces and tight junctions induces a redistribution of F-actinand the tight junction's protein ZO-1, which accompanies the increasedparacellular permeability (Drug Deliv. Rev. 2001; 50:S91-S101). It issuggested that an interaction between chitosan and the tight junctionprotein ZO-1, leads to its translocation to the cytoskeleton.

After removal of the incubated nanoparticles, a gradual increase in TEERvalues was noticed. This phenomenon indicated that the intercellulartight junctions of Caco-2 cell monolayers started to recover gradually;however, the TEER values did not recover to their initial values (FIG.6). Kotzé et al. reported that complete removal of a CS-derived polymer,without damaging the cultured cells, was difficult due to the highlyadhesive feature of CS (Pharm. Res. 1997; 14:1197-1202). This might bethe reason why the TEER values did not recover to their initial values.In contrast, the TEER values of Caco-2 cell monolayers incubated withthe nanoparticles with a negative surface charge (γ-PGA dominated on thesurface, 0.10% γ-PGA:0.01% CS and 0.20% γ-PGA:0.01% CS, FIG. 6) showedno significant differences as compared to the control group (p>0.05).This indicated that γ-PGA does not have any effects on the opening ofthe intercellular tight junctions.

FIG. 8 shows an illustrative protein transport mechanism through acellular layer, including transcellular transport and paracellulertransport. FIG. 9 shows a schematic illustration of a paracellulartransport mechanism. The transcellular protein or peptide transport maybe an active transport or a passive transport mode whereas theparacellular transport is basically a passive mode. Ward et al. reportedand reviewed current knowledge regarding the physiological regulation oftight junctions and paracellular permeability (PSTT 2000; 3:346-358).Chitosan as nanoparticle vehicles for oral delivery of protein drugsavoids the enzymatic inactivation in the gastrointestinal conduit. Thechitosan component of the present nanoparticles has a special feature ofadhering to the mucosal surface and transiently opening the tightjunctions between epithelial cells.

EXAMPLE NO. 7

fCS-γ-PGA Nanoparticle Preparation and CLSM Visualization

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles (FIG. 10)were prepared for the confocal laser scanning microscopy (CLSM) study.The nanoparticles of the present invention display a structure of aneutral polyelectrolyte-complex core surrounded by a positively chargedchitosan shell. Synthesis of the FITC-labeled low-MW CS (fCS) was basedon the reaction between the isothiocyanate group of FITC and the primaryamino groups of CS as reported in the literature (Phamm. Res. 2003;20:1812-1819). Briefly, 100 mg of FITC in 150 ml of dehydrated methanolwere added to 100 ml of 1% low-MW CS in 0.1M acetic acid. After 3 hoursof reaction in the dark at ambient conditions, fCS was precipitated byraising the pH to about 8-9 with 0.5M NaOH. To remove the unconjugatedFITC, the precipitate was subjected to repeated cycles of washing andcentrifugation (40,000×g for 10 min) until no fluorescence was detectedin the supernatant. The fCS dissolved in 80 ml of 0.1M acetic acid wasthen dialyzed for 3 days in the dark against 5 liters of distilledwater, with water replaced on a daily basis. The resultant fCS waslyophilized in a freeze dryer. The fCS-γ-PGA nanoparticles were preparedas per the procedure described in Example No. 4.

Subsequently, the transport medium containing fCS-γ-PGA nanoparticles(0.2 mg/ml) was introduced into the donor compartment of Caco-2 cells,which were pre-cultured on the transwell for 18-21 days. Theexperimental temperature was maintained at 37° C. by a temperaturecontrol system (DH-35 Culture Dish Heater, Warner Instruments Inc.,Hamden, Conn.). After incubation for specific time intervals, testsamples were aspirated. The cells were then washed twice with pre-warmedPBS solution before they were fixed in 3.7% paraformaldehyde (Pharm.Res. 2003; 20:1812-1819). Cells were examined under an inversed CLSM(TCS SL, Leica, Germany). The fluorescence images were observed using anargon laser (excitation at 488 nm, emission collected at a range of510-540 nm).

CLSM was used to visualize the transport of the fluorescence-labeledCS-γ-PGA (fCS-γ-PGA) nanoparticles across the Caco-2 cell monolayers.This non-invasive method allows for optical sectioning and imaging ofthe transport pathways across the Caco-2 cell monolayers, withoutdisrupting their structures (J. Control. Release 1996; 39:131-138).FIGS. 7 a and 7 b show the fluorescence images of 4 optical sections ofa Caco-2 cell monolayer that had been incubated with the fCS-γ-PGAnanoparticles having a positive surface charge (0.10% γ-PGA:0.20% CS,zeta potential: about 21 mV) for 20 and 60 min, respectively. As shown,after 20 min of incubation with the nanoparticles, intense fluorescencesignals at intercellular spaces were observed at depths of 0 and 5 μmfrom the apical (upper) surface of the cell monolayer. The intensity offluorescence became weaker at levels deeper than 10 μm from the apicalsurface of the cell monolayer and was almost absent at depths ≧15 μm(FIG. 7 a).

After 60 minutes of incubation with the nanoparticles, the intensity offluorescence observed at intercellular spaces was stronger and appearedat a deeper level than those observed at 20 min after incubation. Theseobservations confirmed with our TEER results that the nanoparticles witha positive surface charge (CS dominated on the surface) were able toopen the tight junctions between Caco-2 cells and allowed transport ofthe nanoparticles by passive diffusion via the paracellular pathways.

EXAMPLE NO. 8

In Vivo Study with Fluorescence-labeled Nanoparticles

Fluorescence (FITC)-labeled CS-γ-PGA (fCS-γ-PGA) nanoparticles wereprepared for the confocal laser scanning microscopy (CLSM) study. Afterfeeding rats with fCS-γ-PGA nanoparticles, the rats are sacrificed at apre-determined time and the intestine is isolated for CLSM examination.The fluorescence images of the nanoparticles were clearly observed byCLSM that penetrates through the mouse intestine at appropriate time andat various depths from the inner surface toward the exterior surface ofthe intestine, including duodenum, jejunum, and ileum.

EXAMPLE NO. 9

Insulin Loading Capacity in Nanoparticles

Fluorescence (FITC)-labeled γ-PGA was added into chitosan solution toprepare fluorescence (FITC)-labeled, insulin-loaded CS-γ-PGAnanoparticles for in vivo animal study with confocal laser scanningmicroscopy (CLSM) assessment and bioactivity analysis. Theinsulin-loaded CS-γ PGA nanoparticles are by using the ionic-gelationmethod upon addition of insulin mixed with γ-PGA solution into CSsolution, followed by magnetic stirring in a container.

Model insulin used in the experiment and disclosed herein is obtainedfrom bovine pancreas (Sigma-Aldrich, St. Louis, Mo.), having a molecularformula of C₂₅₄H₃₇₇N₆₅O₇₅S₆ with a molecular weight of about 5733.5 andan activity of ≧27 USP units/mg. The insulin contains two-chainpolypeptide hormone produced by the β-cells of pancreatic islets. The αand β chains are joined by two interchain disulfide bonds. Insulinregulates the cellular uptake, utilization, and storage of glucose,amino acids, and fatty acids and inhibits the breakdown of glycogen,protein, and fat. The insulin from Sigma-Aldrich contains about 0.5%zinc. Separately, insulin can be obtained from other sources, such ashuman insulin solution that is chemically defined, recombinant fromSaccharomyces cerevisiae. Some aspects of the invention relate tonanoparticles with insulin in the core, wherein the insulin may containintermediate-acting, regular insulin, rapid-acting insulin,sustained-acting insulin that provides slower onset and longer durationof activity than regular insulin, or combinations thereof.

Examples of insulin or insulin analog products include, but not limitedto, Humulin® (by Eli Lilly), Humalog® (by Eli Lilly) and Lantus® (byAventis), and Novolog® Mix70/30 (by Novo Nordisk). Humalog (insulinlispro, rDNA origin) is a human insulin analog that is a rapid-acting,parenteral blood glucose-lowering agent. Chemically, it is Lys(B28),Pro(B29) human insulin analog, created when the amino acids at positions28 and 29 on the insulin B-chain are reversed. Humalog is synthesized ina special non-pathogenic laboratory strain of Escherichia coli bacteriathat has been genetically altered by the addition of the gene forinsulin lispro. Humalog has the empirical formula C₂₅₇H₃₈₃N₆₅O₇₇S₆ and amolecular weight of 5808, identical to that of human insulin. The vialsand cartridges contain a sterile solution of Humalog for use as aninjection. Humalog injection consists of zinc-insulin lispro crystalsdissolved in a clear aqueous fluid. Each milliliter of Humalog injectioncontains insulin lispro 100 Units, 16 mg glycerin, 1.88 mg dibasicsodium phosphate, 3.15 mg m-cresol, zinc oxide content adjusted toprovide 0.0197 mg zinc ion, trace amounts of phenol, and water forinjection. Insulin lispro has a pH of 7.0-7.8. Hydrochloric acid 10%and/or sodium hydroxide 10% may be added to adjust pH.

Humulin is used by more than 4 million people with diabetes around theworld every day. Despite its name, this insulin does not come from humanbeings. It is identical in chemical structure to human insulin and ismade in a factory using a chemical process called recombinant DNAtechnology. Humulin L is an amorphous and crystalline suspension ofhuman insulin with a slower onset and a longer duration of activity (upto 24 hours) than regular insulin. Humulin U is a crystalline suspensionof human insulin with zinc providing a slower onset and a longer andless intense duration of activity (up to 28 hours) than regular insulinor the intermediate-acting insulins (NPH and Lente).

LANTUS® (insulin glargine [rDNA origin] injection) is a sterile solutionof insulin glargine for use as an injection. Insulin glargine is arecombinant human insulin analog that is a long-acting (up to 24-hourduration of action), parenteral blood-glucose-lowering agent. LANTUS isproduced by recombinant DNA technology utilizing a non-pathogeniclaboratory strain of Escherichia coli (K12) as the production organism.Insulin glargine differs from human insulin in that the amino acidasparagine at position A21 is replaced by glycine and two arginines areadded to the C-terminus of the B-chain. Chemically, it is21^(A)-Gly-30^(B)a-L-Arg-30^(B)b-L-Arg-human insulin and has theempirical formula C₂₆₇H₄₀₄N₇₂O₇₈S₆ and a molecular weight of 6063.

LANTUS consists of insulin glargine dissolved in a clear aqueous fluid.Each milliliter of LANTUS (insulin glargine injection) contains 100 IU(3.6378 mg) insulin glargine. Inactive ingredients for the 10 mL vialare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, 20 mcg polysorbate20, and water for injection. Inactive ingredients for the 3 mL cartridgeare 30 mcg zinc, 2.7 mg m-cresol, 20 mg glycerol 85%, and water forinjection.

Novolog® Mix70/30 (70% insulin aspart protamine suspension and 30%insulin aspart injection [rDNA origin]) is a human insulin analogsuspension. Novolog® Mix70/30 is a blood glucose-lowering agent with arapid onset and an intermediate duration of action. Insulin aspart ishomologous with regular human insulin with the exception of a singlesubstitution of the amino acid praline by aspartic acid in position B28,and is produced by recombinant DNA technology utilizing Saccharomycescerevisiae as the production organism. Insulin aspart (Novolog) has theempirical formula C₂₅₆H₃₈₁N₆₅O₇₉S₆ and a molecular weight of 5826.Novolog® Mix70/30 is a uniform, white sterile suspension that containszinc 19.6 μg/ml and other components.

The nanoparticles with two insulin concentrations are prepared at achitosan to γ-PGA ratio of 0.75 mg/ml to 0.167 mg/ml. Their particlesize and zeta potential are shown in Table 2 below.

TABLE 2 Mean Insulin Conc. Particle Size Polydispersity Index ZetaPotential (mg/ml) (n = 5) (nm) (PI) (mV) 0* 145.6 ± 1.9 0.14 ± 0.01+32.11 ± 1.61 0.042 185.1 ± 5.6 0.31 ± 0.05 +29.91 ± 1.02 0.083 198.4 ±6.2 0.30 ± 0.09 +27.83 ± 1.22 *control reference without insulin

Further, their association efficiency of insulin and loading capacity ofinsulin are analyzed, calculated and shown in FIGS. 11 and 12, accordingto the following formula:

$\begin{matrix}{{Insulin}\mspace{14mu}{Association}} \\{{Efficiency}\mspace{14mu}\left( {{AE}\mspace{14mu}\%} \right)} \\{{Loading}\mspace{14mu}{Capacity}\mspace{14mu}({LC})}\end{matrix}\begin{matrix}{= {\frac{\left( {{{Total}\mspace{14mu}{amount}{\mspace{11mu}\;}{of}\mspace{14mu}{insulin}} - {{Insulin}\mspace{14mu}{in}\mspace{14mu}{supernatant}}} \right)}{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{insulin}} \times 100\%}} \\{= {\frac{\left( {{{Total}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{insulin}} - {{Insulin}\mspace{14mu}{in}\mspace{14mu}{supernatant}}} \right)}{{Weight}{\mspace{11mu}\;}{of}\mspace{14mu}{recovered}\mspace{14mu}{particles}} \times 100\%}}\end{matrix}$

FIG. 11 shows loading capacity and association efficiency of insulin innanoparticles of chitosan and γ-PGA, whereas FIG. 12 shows loadingcapacity and association efficiency of insulin in nanoparticles ofchitosan alone (in absence of γ-PGA) as reference. The data clearlydemonstrates that both the insulin loading capacity and insulinassociation efficiency are statistically higher for the nanoparticleswith γ-PGA in the core. The AE (40˜55%) and LC (5.0˜14.0%) of insulinfor CS-γ PGA nanoparticles was obtained by using ionic-gelation methodupon addition of insulin mixed with γ-PGA solution into CS solution,followed by magnetic stirring for nanoparticle separation. Some aspectsof the invention relate to an oral dose of nanoparticles thateffectively enhance intestinal or blood brain paracellular transportcomprising a negative component (such as γ-PGA, α-PGA, PGA derivatives,heparin, or alginate) in the core and low molecular weight chitosan,wherein the chitosan dominates on a surface of the nanoparticles withpositive charges.

EXAMPLE NO. 10

Insulin Loading in PGA Nanoparticles

The nanoparticles with two core substrates (γ-PGA and α-PGA) areprepared at a chitosan to PGA ratio of 0.75 mg/ml to 0.167 mg/ml withinsulin concentration at 0.083 mg/ml (sample size, n=3). Their particlesize, zeta potential, and insulin loading efficiency are quitecomparable and are shown in Table 3 below.

TABLE 3 Loading Core Mean Particle Polydispersity Zeta PotentialEfficiency Substrate Size (nm) Index (PI) (mV) (%) γ-PGA 218.4 ± 5.20.32 ± 0.09 +25.4 ± 1.22 52.1 ± 4.3 α-PGA 207.6 ± 9.3 0.29 ± 0.07 +26.8± 1.01 59.1 ± 5.0

Some aspects of the invention relate to a dose of nanoparticles thateffectively enhance intestinal or blood brain paracellular transportcomprising a polyanionic component (such as γ-PGA, α-PGA, PGAderivatives, heparin, heparin analogs, low molecular weight heparin,glycosaminoglycans, or alginate) in the core and low molecular weightchitosan in the shell, wherein the chitosan dominates on a surface ofthe nanoparticles with surface positive charges. In use, firstly,encapsulate the Alzheimer's drug in the chitosan shell nanoparticle asdescribed herein, wherein the nanoparticle is partially crosslinked(optionally) to enhance its biodurability. Then intra-venously injectthe nanoparticles, whereby the nanoparticles pass to the brain in bloodcirculation. The chitosan shell of the nanoparticles adheres to thesurface adjacent the tight junction in the brain. Thereafter, thechitosan nanoparticle opens the tight junction, wherein the Alzheimer'sdrug is released after passing the tight junction for therapeutictreatment. In one embodiment, the nanoparticles are in a spherical shapehaving a mean particle size of about 50 to 250 nanometers, preferably150 nanometers to 250 nanometers.

In one example, intravenous administration of the nanoparticlescomprising chitosan shell substrate, polyanionic core substrate and atleast one bioactive agent for treating Alzheimer's disease in a patientis typically performed with 10 mg to 40 mg of active agent per day overa period of one month to one year. The bioactive agent is selected froma group consisting of donepezile, rivastignine, galantamine, and/orthose trade-named products, such as memantine hydrochloride (Axura® byMerz Pharmaceuticals), donepezil hydrochloride (Aricept® by Eisai Co.Ltd.), rivastigmine tartrate (Exelon® by Novartis), galantaminehydrochloride (Reminyl® by Johnson & Johnson), and tacrine hydrochloride(Cognex® by Parke Davis).

Some aspects of the invention relate to a nanoparticle with a coresubstrate comprising polyglutamic acids such as water soluble salt ofpolyglutamic acids (for example, ammonium salt) or metal salts ofpolyglutamic acid (for example, lithium salt, sodium salt, potassiumsalt, magnesium salt, and the like). In one embodiment, the form ofpolyglutamic acid may be selected from a group consisting ofpoly-α-glutamic acid, poly-L-α-glutamic acid, poly-γ-glutamic acid,poly-D-glutamic acid, poly-γ-D-glutamic acid, poly-γ-DL-glutamic acid,poly-L-glutamic acid (manufactured by Sigma-Aldrich, St. Louis, Mo.),and PEG or PHEG derivatives of polyglutamic acid. Alginate is generallynon-biodegradable; however, it is stipulated that an alginate particlewith about 30-50 kDa molecular weight is kidney inert. Heparin withnegatively charged side-groups has a general chemical structure as shownbelow:

Some aspects of the invention relate to the negatively chargedglycosaminoglycans (GAGs) as the core substrate of the presentnanoparticles. GAGs may be used to complex with a low-molecular-weightchitosan to form drug-carrier nanoparticles. GAGs may also conjugatewith the proteins (for example, monoclonal antibodies) as disclosedherein to enhance the bonding efficiency of the core substrate in thenanoparticles. Particularly, the negatively charged core substrate (suchas GAGs, heparin, PGA, alginate, and the like) of the nanoparticles ofthe present invention may conjugate with chondroitin sulfate, hyaluronicacid, PDGF-BB, BSA, EGF, MK, VEGF, KGF, bFGF, aFGF, MK, PTN, etc.

Calceti et al. reported an in vivo evaluation of an oral insulin-PEGdelivery system (Eur J Pharma Sci 2004; 22:315-323). Insulin-PEG wasformulated into mucoadhesive tablets constituted by the thiolatedpolymer poly(acrylic acid)-cysteine. The therapeutic agent was sustainedreleased from these tablets within 5 hours. In vivo, by oraladministration to diabetic mice, the glucose levels were found todecrease significantly over the time. Further, Krauland et al. reportedanother oral insulin delivery study of thiolated chitosan-insulintablets on non-diabetic rats (J. Control. Release 2004, 95:547-555). Thedelivery tablets utilized 2-Iminothiolane covalently linked to chitosanto form chitosan-TBA (chitosan-4-thiobutylamidine) conjugate. After oraladministration of chitosan-TBA-insulin tablets to non-diabetic consciousrats, the blood glucose level decreased significantly for 24 hours;supporting the observation of sustained insulin release of the presentlydisclosed nanoparticles herein through intestinal absorption. In afurther report by Morcol et al. (Int. J. Pharm. 2004; 277:91-97), anoral delivery system comprising calcium phosphate-PEG-insulin-caseinparticles displays a prolonged hypoglycemic effect after oraladministration to diabetic rats.

Pan et al. disclosed chitosan nanoparticles improving the intestinalabsorption of insulin in vivo (Int J Pharma 2002; 249:139-147) withinsulin-chitosan nanoparticles at a particle size of 250-400 nm, apolydispersity index smaller than 0.1, positively charged and stable.After administering the insulin-chitosan nanoparticles, it was foundthat the hypoglycemic was prolonged with enhanced pharmacologicalbioavailability. Their data confirmed our observation as shown in FIGS.11 and 12; however, the insulin loading capacity and insulin associationefficiency of the present invention are substantially higher for thechitosan-insulin nanoparticles with γ-PGA in the core as the coresubstrate.

EXAMPLE NO. 11

Insulin Nanoparticle Stability

FIG. 13 shows the stability of insulin-loaded nanoparticles of thepresent invention with an exemplary composition of CS 0.75 mg/ml, γ-PGA0.167 mg/ml, and insulin 0.083 mg/ml. The prepared insulin-loadednanoparticles suspended in deionized water are stable during storage upto 40 days. First (in FIG. 13), the insulin content in the nanoparticlestorage solution maintains at about a constant level of 9.5%. Thenanoparticle stability is further evidenced by the substantiallyconstant particle size at about 200 nm and substantially constant zetapotential of about +28 mV over the period of about 40 days. It iscontemplated that the insulin-containing nanoparticles of the presentinvention would further maintain their biostability when formulated in asoft gelcap configuration that further isolates the nanoparticles fromenvironmental effects, such as sunlight, heat, air conditions, and thelike. Some aspects of the invention provide a gelcap pill containing adosage of insulin nanoparticles effective amount of the insulin to treator manage the diabetic patients, wherein the stability of theinsulin-containing nanoparticles is at least 40 days, preferably morethan 6 months, and most preferably more than a couple of years. By“effective amount of the insulin”, it is meant that a sufficient amountof insulin will be present in the dose to provide for a desiredtherapeutic, prophylatic, or other biological effect when thecompositions are administered to a host in the single dosage forms.

Thus, for convenient and effective oral administration, pharmaceuticallyeffective amounts of the nanoparticles of this invention can betabletted with one or more excipient, encased in capsules such as gelcapsules, and suspended in a liquid solution and the like. Thenanoparticles can be suspended in a deionized solution or the like forparenteral administration. The nanoparticles may be formed into a packedmass for ingestion by conventional techniques. For instance, thenanoparticles may be encapsulated as a “hard-filled capsule” or a“soft-elastic capsule” using known encapsulating procedures andmaterials. The encapsulating material should be highly soluble ingastric fluid so that the particles are rapidly dispersed in the stomachafter the capsule is ingested. Each unit dose, whether capsule ortablet, will preferably contain nanoparticles of a suitable size andquantity that provides pharmaceutically effective amounts of thenanoparticles. The applicable shapes and sizes of soft gel capsules mayinclude round, oval, oblong, tube or suppository shape with sizes from0.75 mm to 80 mm or larger. The volume of the capsules can be from 0.05cc to more than 5 cc.

EXAMPLE NO. 12

In Vitro Insulin Release Study

FIG. 14 show a representative protein drug (for example, insulin)release profile in a pH-adjusted solution for pH-sensitivity study withan exemplary composition of CS 0.75 mg/ml, γ-PGA 0.167 mg/ml, andinsulin 0.083 mg/ml in nanoparticles. In one embodiment, the exemplarycomposition may include each component at a concentration range of ±10%as follows: CS 0.75 mg/ml (a concentration range of 0.67 to 0.83 mg/ml),γ-PGA 0.167 mg/ml (a concentration range of 0.150 to 0.184 mg/ml), andinsulin 0.083 mg/ml (a concentration range of 0.075 to 0.091 mg/ml).First, solution of the insulin-loaded nanoparticles was adjusted to pH2.5 to simulate the gastric environment in a DISTEK-2230A container at37° C. and 100 rpm. Samples (n=5) were taken at a pre-determinedparticular time interval and the particle-free solution was obtained bycentrifuging at 22,000 rpm for 30 minutes to analyze the free orreleased insulin in solution by HPLC.

Until the free insulin content in the sample solution approaches aboutconstant of 26% (shown in FIG. 14), the pH was adjusted to 6.6 tosimulate the entrance portion of the intestine. The net released insulinduring this particular time interval is about (from 26% to 33%) 7%. Inother words, the nanoparticles are quite stable (evidenced by minimalmeasurable insulin in solution) for both the pH 2.5 and pH 6.6 regions.To further simulate the exit portion of the intestine, theinsulin-containing nanoparticle solution is adjusted to pH 7.4. Theremaining insulin (about 67%) is released from the nanoparticles. Asdiscussed above, the insulin in nanoparticles would be more effective topenetrate the intestine wall in paracellular transport mode than thefree insulin because of the nanoparticles of the present invention withchitosan at the outer surface (preferential mucosal adhesion on theintestinal wall) and positive charge (enhancing paracellular tightjunction transport).

EXAMPLE NO. 13

In Vivo Study with Insulin-loaded Fluorescence-labeled Nanoparticles

In the in vivo study, rats were injected with streptozotocin (STZ 75mg/kg intraperitoneal) in 0.01M citrate buffer (pH 4.3) to inducediabetes rats. The blood from the rat's tail was analyzed with acommercially available glucometer for blood glucose. The blood glucoselevel on Wistar male rats at no fasting (n=5) is measured at 107.2±8.1mg/dL for normal rats while the blood glucose level is at 469.7±34.2mg/dL for diabetic rats. In the animal study, diabetic rats were fastingfor 12 hours and subjected to four different conditions: (a) oraldeionized water (DI) administration; (b) oral insulin administration at30 U/kg; (c) oral insulin-loaded nanoparticles administration at 30U/kg; and (d) subcutaneous (SC) insulin injection at 5 U/kg as positivecontrol. The blood glucose concentration from rat's tail was measuredover the time in the study.

FIG. 15 shows glucose change (hypoglycemic index) versus time of the invivo animal study (n=5). The glucose change as a percentage of baselines for both oral DI administration and oral insulin administrationover a time interval of 8 hours appears relatively constant within theexperimental measurement error range. It is illustrative thatsubstantially all insulin from the oral administration route has beendecomposed in rat stomach. As anticipated, the glucose decrease for theSC insulin injection route appears in rat blood in the very early timeinterval and starts to taper off after 3 hours in this exemplary study.

The most important observation of the study comes from the oraladministration route with insulin-loaded nanoparticles. The bloodglucose begins to decrease from the base line at about 2 hours afteradministration and sustains at a lower glucose level at more than 8hours into study. It implies that the current insulin-loadednanoparticles may modulate the glucose level in animals in a sustainedor prolonged effective mode. Some aspects of the invention provide amethod of treating diabetes of a patient comprising orally administeringinsulin-containing nanoparticles with a dosage effective amount of theinsulin to treat the diabetes, wherein at least a portion of thenanoparticles comprises a positively charged shell substrate and anegatively charged core substrate. In one embodiment, the dosageeffective amount of the insulin to treat the diabetes comprises aninsulin amount of between about 15 units to 45 units per kilogram bodyweight of the patient, preferably 20 to 40 units, and most preferably atabout 25 to 35 units insulin per kilogram body weight.

Some aspects of the invention relate to a novel nanoparticle system thatis composed of a low-MW CS and γ-PGA with CS dominated on the surfacesbeing configured to effectively open the tight junctions between Caco-2cell monolayers. The surface of the nanoparticles is characterized witha positive surface charge. In one embodiment, the nanoparticles of theinvention enables effective intestinal delivery for bioactive agent,including peptide, polypeptide, protein drugs, other large hydrophilicmolecules, and the like. Such polypeptide drugs can be any natural orsynthetic polypeptide that may be orally administered to a humanpatient.

Exemplary drugs of the present invention include, but are not limitedto, insulin; growth factors, such as epidermal growth factor (EGF),insulin-like growth factor (IGF), transforming growth factor (TGF),nerve growth factor (NGF), platelet-derived growth factor (PDGF), bonemorphogenic protein (BMP), fibroblast growth factor and the like;somatostatin; somatotropin; somatropin; somatrem; calcitonin;parathyroid hormone; colony stimulating factors (CSF); clotting factors;tumor necrosis factors: interferons; interleukins; gastrointestinalpeptides, such as vasoactive intestinal peptide (VIP), cholecytokinin(CCK), gastrin, secretin, and the like; erythropoietins; growth hormoneand GRF; vasopressins; octreotide; pancreatic enzymes; dismutases suchas superoxide dismutase; thyrotropin releasing hormone (TRH); thyroidstimulating hormone; luteinizing hormone; LHRH; GHRH; tissue plasminogenactivators; macrophage activator; chorionic gonadotropin; heparin;atrial natriuretic peptide; hemoglobin; retroviral vectors; relaxin;cyclosporin; oxytocin; vaccines; monoclonal antibodies; and the like;and analogs and derivatives of these compounds.

The bioactive agent of the present invention may be selected from groupconsisting of oxytocin, vasopressin, adrenocorticotrophic hormone,prolactin, luliberin or luteinising hormone releasing hormone, growthhormone, growth hormone releasing factor, somatostatin, glucagon,interferon, gastrin, tetragastrin, pentagastrin, urogastroine, secretin,calcitonin, enkephalins, endorphins, angiotensins, renin, bradykinin,bacitracins, polymixins, colistins, tyrocidin, gramicidines, andsynthetic analogues, modifications and pharmacologically activefragments thereof, monoclonal antibodies and soluble vaccines.

In another embodiment, the nanoparticles of the invention increase theabsorption of bioactive agents across the blood brain barrier and/or thegastrointestinal barrier. In still another embodiment, the nanoparticleswith chitosan at an outer layer and surface positive charge serve as anenhancer in enhancing paracellular drug (bioactive agent) transport ofan administered bioactive agent when the bioactive agent andnanoparticles are orally administrated in a two-component system, ororally administered substantially simultaneously.

EXAMPLE NO. 14

Paracellular Transport and Enhancers

Chitosan and its derivatives may function as intestinal absorptionenhancers (that is, paracellular transport enhancers). Chitosan, whenprotonated at an acidic pH, is able to increase the paracellularpermeability of peptide drugs across mucosal epithelia. Some aspects ofthe invention provide co-administration of nanoparticles of the presentinvention and at least one paracellular transport enhancer (innon-nanoparticle form or nanoparticle form). In one embodiment, thenanoparticles can be formulated by co-encapsulation of the at least oneparacellular transport enhancer and at least one bioactive agent,optionally with other components. The enhancer may be selected from thegroup consisting of Ca²⁺ chelators, bile salts, anionic surfactants,medium-chain fatty acids, phosphate esters, and chitosan or chitosanderivatives. In one embodiment, the nanoparticles of the presentinvention comprises a positively charged shell substrate and anegatively charged core substrate, for example, nanoparticles composedof γ-PGA and chitosan that is characterized with a substantiallypositive surface charge.

In some embodiment, the nanoparticles of the present invention and theat least one paracellular transport enhancer are encapsulated in a softgel, pill, or enteric coated. The enhancers and the nanoparticles wouldarrive at the tight junction about the same time for enhancing openingthe tight junction. In another embodiment, the at least one paracellulartransport enhancer is co-enclosed within the shell of the nanoparticlesof the present invention. Therefore, some broken nanoparticles wouldrelease enhancers to assist the nanoparticles to open the tightjunctions of the epithelial layers. In an alternate embodiment, the atleast one enhancer is enclosed within a second nanoparticle havingpositive surface charges, particularly a chitosan type nanoparticle.When the drug-containing first nanoparticles of the present inventionare co-administered with the above-identified second nanoparticlesorally, the enhancers within the second nanoparticles are released inthe intestinal tract to assist the drug-containing first nanoparticlesto open and pass the tight junction.

EXAMPLE NO. 15

Nanoparticles with Complexed Calcitonin

Calcitonin is a protein drug that serves therapeutically as calciumregulators for treating osteoporosis (J. Pharm. Pharmacol. 1994;46:547-552). Calcitonin has a molecular formula of C₁₄₅H₂₄₀N₄₄O₄₈S₂ witha molecular weight of about 3431.9 and an isoelectric point of 8.7. Thenet charge for calcitonin at pH7.4 is positive that is suitable tocomplex or conjugate with negatively charged core substrate, such asγ-PGA or α-PGA. In preparation, nanoparticles were obtained uponaddition of a mixture of γ-PGA plus calcitonin aqueous solution (pH 7.4,2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®, BrandTech ScientificInc., Germany), into a low-MW CS aqueous solution (pH 6.0, 10 ml) atconcentrations higher than 0.10% by w/v under magnetic stirring at roomtemperature to ensure positive surface charge. Nanoparticles werecollected by ultracentrifugation at 38,000 rpm for 1 hour. Calcitonin istotally or substantially totally encapsulated in the nanoparticles.Supernatants were discarded and nanoparticles were resuspended indeionized water as the solution products, further encapsulated in softgels or further treated with an enteric coating.

EXAMPLE NO. 16

Nanoparticles with Conjugated Vancomycin

Vancomycin is a protein drug that serves therapeutically as antibioticagainst bacterial pathogens. Vancomycin has a molecular formula ofC₆₆H₇₅N₉O₂₄ with a molecular weight of about 1485.7 and an isoelectricpoint of 5.0. The net charge for vancomycin at pH7.4 is negative that issuitable to complex or conjugate with a portion of negatively chargedshell substrate, such as chitosan. In preparation, nanoparticles wereobtained upon addition of a mixture of γ-PGA plus vancomycin aqueoussolution (pH 7.4, 2 ml), using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a low-MW CS aqueous solution(pH 6.0, 10 ml) with excess concentrations under magnetic stirring atroom temperature, wherein CS concentration is provided sufficiently toconjugate vancomycin, to counterbalance γ-PGA, and exhibit positivesurface charge for the nanoparticles. Nanoparticles were collected byultracentrifugation at 38,000 rpm for 1 hour. Vancomycin is wholly orsubstantially totally encapsulated in the nanoparticles. Supernatantswere discarded and nanoparticles were resuspended in deionized water asthe solution products, further encapsulated in soft gels or furthertreated with an enteric coating.

Some aspects of the invention relate to a method of enhancing intestinalor blood brain paracellular transport of bioactive agents configured andadapted for delivering at least one bioactive agent in a patientcomprising administering nanoparticles composed of γ-PGA and chitosan,wherein the nanoparticles are loaded with a therapeutically effectiveamount or dose of the at least one bioactive agent. The nanoparticle ofthe present invention is an effective intestinal delivery system forpeptide and protein drugs and other large hydrophilic molecules. In afurther embodiment, the bioactive agent is selected from the groupconsisting of proteins, peptides, nucleosides, nucleotides, antiviralagents, antineoplastic agents, antibiotics, and anti-inflammatory drugs.In a further embodiment, the bioactive agent is selected from the groupconsisting of calcitonin, cyclosporin, insulin, oxytocin, tyrosine,enkephalin, tyrotropin releasing hormone (TRH), follicle stimulatinghormone (FSH), luteinizing hormone (LH), vasopressin and vasopressinanalogs, catalase, superoxide dismutase, interleukin-II (IL2),interferon, colony stimulating factor (CSF), tumor necrosis factor (TNF)and melanocyte-stimulating hormone. In a further embodiment, thebioactive agent is an Alzheimer antagonist.

EXAMPLE NO. 17

Nanoparticles with Heparin Core Substrate

Heparin is a negatively charged drug that serves therapeutically asanti-coagulant. Heparin is generally administered by intravenousinjection. Some aspects of the invention relate to heparin nanoparticlesfor oral administration or subcutaneous administration. In a furtherembodiment, heparin serves as at least a portion of the core substratewith chitosan as shell substrate, wherein heparin conjugate at least onebioactive agent as disclosed herein. In preparation, nanoparticles wereobtained upon addition of heparin Leo aqueous solution (2 ml), using apipette (0.5-5 ml, PLASTIBRAND®, BrandTech Scientific Inc., Germany),into a low-MW CS aqueous solution (pH 6.0, 10 ml) with excessconcentrations under magnetic stirring at room temperature.Nanoparticles were collected by ultracentrifugation at 38,000 rpm for 1hour. Heparin is totally or substantially totally encapsulated in thenanoparticles. In other words, heparin is substantially maintainedwithin the intact nanoparticles during the nanoparticle delivery phaseorally. Thus, heparin does not cause any significant effect until thenanoparticles dissociate or biodegrade to release the core contents.Table 4 shows the conditions of solution preparation and the averagenanoparticle size.

TABLE 4 Chitosan conc. Conditions Heparin conc. @2 ml @10 ml Particlesize (nm) A 200 iu/ml 0.09% 298.2 ± 9.3 B 100 iu/ml 0.09% 229.1 ± 4.5 C 50 iu/ml 0.09% 168.6 ± 1.7 D  25 iu/ml 0.09% 140.1 ± 2.3

To evaluate the pH stability of the heparin-containing nanoparticlesfrom Example no. 17, the nanoparticles from Condition D in Table 4 aresubjected to various pH for 2 hours (sample size=7). Table 5 shows theaverage size, size distribution (polydispersity index: PI) and zetapotential (Zeta) of the nanoparticles at the end of 2 hours undervarious pH environments. The data shows the nanoparticles are relativelystable. In one embodiment, the nanoparticles of the present inventionmay include heparin, heparin sulfate, small molecular weight heparin,and heparin derivatives.

TABLE 5 pH 1.5 2.6 6.6 7.4 Deionized water @5.9 Size (nm) 150 ± 9  160 ±12  153 ± 2  154 ± 4  147 ± 5  PI 0.54 ± 0.03 0.50 ± 0.04 0.08 ± 0.020.32 ± 0.03 0.37 ± 0.02 Zeta(+) 15 ± 2  33 ± 6   15 ± 0.1  11 ± 0.2 18 ±4 

In a further embodiment, a growth factor such as bFGF withpharmaceutically effective amount is added to heparin Leo aqueoussolution before the pipetting step in Example No. 16. In our laboratory,growth factors and proteins with pharmaceutically effective amount havebeen successfully conjugated with heparin to form nanoparticles of thepresent invention with chitosan as the shell substrate, wherein thegrowth factor is selected from the group consisting of VascularEndothelial Growth Factor (VEGF), Vascular Endothelial Growth Factor 2(VEGF2), basic Fibroblast Growth Factor (bFGF), Vascular EndothelialGrowth Factor 121 (VEGF121), Vascular Endothelial Growth Factor 165(VEGF165), Vascular Endothelial Growth Factor 189 (VEGF189), VascularEndothelial Growth Factor 206 (VEGF206), Platelet Derived Growth Factor(PDGF), Platelet Derived Angiogenesis Factor (PDAF), Transforming GrowthFactor-β (TGF-β), Transforming Growth Factor-α (TGF-α), Platelet DerivedEpidermal Growth Factor (PDEGF), Platelet Derived Wound Healing Formula(PDWHF), epidermal growth factor, insulin-like growth factor, acidicFibroblast Growth Factor (aFGF), human growth factor, and combinationsthereof; and the protein is selected from the group consisting ofhaemagglutinin (HBHA), Pleiotrophin, buffalo seminal plasma proteins,and combinations thereof.

In a co-pending application, U.S. patent application Ser. No. 10/916,170filed Aug. 11, 2004, it is disclosed that a biomaterial with free aminogroups of lysine, hydroxylysine, or arginine residues within biologictissues is crosslinkable with genipin, a crosslinker (Biomaterials 1999;20:1759-72). It is also disclosed that the crosslinkable biomaterial maybe crosslinked with a crosslinking agent or with light, such asultraviolet irradiation, wherein the crosslinkable biomaterial may beselected from the group consisting of collagen, gelatin, elastin,chitosan, NOCC(N, O, carboxylmethyl chitosan), fibrin glue, biologicalsealant, and the like. Further, it is disclosed that a crosslinkingagent may be selected from the group consisting of genipin, itsderivatives, analog (for example, aglycon geniposidic acid),stereoisomers and mixtures thereof. In one embodiment, the crosslinkingagent may further be selected from the group consisting of epoxycompounds, dialdehyde starch, glutaraldehyde, formaldehyde, dimethylsuberimidate, carbodiimides, succinimidyls, diisocyanates, acyl azide,reuterin, ultraviolet irradiation, dehydrothermal treatment,tris(hydroxymethyl)phosphine, ascorbate-copper, glucose-lysine andphoto-oxidizers, and the like.

In one embodiment, it is disclosed that loading drug onto achitosan-containing biological material crosslinked with genipin orother crosslinking agent may be used as biocompatible drug carriers fordrug slow-release or sustained release. Several biocompatible plasticpolymers or synthetic polymers have one or more amine group in theirchemical structures, for example poly(amides) or poly(ester amides). Theamine group may become reactive toward a crosslinking agent, such asglutaraldehyde, genipin or epoxy compounds of the present invention. Inone embodiment, the nanoparticles comprised of crosslinkable biomaterialis crosslinked, for example up to about 50% degree or more ofcrosslinking, preferably about 1 to about 20% degree of crosslinking ofthe crosslinkable components of the biomaterial, enabling sustainedbiodegradation of the biomaterial and/or sustained drug release.

By modifying the chitosan structure to alter its charge characteristics,such as grafting the chitosan with methyl, alkyl (for example, ethyl,propyl, butyl, isobutyl, etc.), polyethylene glycol (PEG), or heparin(including low molecular weight heparin, regular molecular weightheparin, and genetically modified heparin), the surface charge density(zeta potential) of the CS-γ PGA nanoparticles may become more pHresistant or hydrophilic. In one embodiment, the chitosan is graftedwith polyacrylic acid or a polymer with a chemical formula:

where R is ≧12

By way of illustration, trimethyl chitosan chloride might be used informulating the CS-γ PGA nanoparticles for maintaining its sphericalbiostability at a pH lower than pH 2.5, preferably at a pH as low as1.0. Some aspects of the invention provide a drug-loadedchitosan-containing biological material crosslinked with genipin orother crosslinking agent as a biocompatible drug carrier for enhancingbiostability at a pH lower than pH 2.5, preferably within at a pH as lowas 1.0.

FIG. 16 shows an illustrative mechanism of nanoparticles released fromthe enteric coating. FIG. 16(A) shows the phase of nanoparticles in thegastric cavity, wherein the nanoparticles 82 are encapsulated within aninitial enteric coating 81. FIG. 16(B) shows a schematic of the coatednanoparticles during the phase of entering small intestine, wherein theenteric coating starts to dissolve 83 and a portion of nanoparticles 83starts to release. FIG. 16(C) shows the phase of nanoparticles in theintestinal tract, wherein the nanoparticles open the tight junctions asdescribed above. In an alternate embodiment, nanoparticles may bereleased from alginate-calcium coating. In preparation, nanoparticlesare first suspended in a solution that contains calcium chloride,wherein the calcium ions are positively charged. With a pipette,alginate with negatively charged carboxyl groups is slowly added to thecalcium chloride solution. Under gentle stirring, the alginate-calciumstarts to conjugate, gel, and coat on the nanoparticle surface. Insimulated oral administration of the alginate-calcium coatednanoparticles, nanoparticles start to separate from the coating whenthey enter the small intestines.

It is known that Zn (zinc) is usually added in the biosynthesis andstorage of insulin. FIGS. 17 and 18 show a schematic of insulinconjugated with a polyanionic compound (i.e., γ-PGA in this case) via Znand thus increase its loading efficiency and loading content in thenanoparticles of the present invention. It is further demonstrated thatZn may complex with the histidine and glutamic acid residues in insulinto increase the insulin stability and enhance controlled releasecapability or sustained therapy. Some aspects of the invention relate toa nanoparticle characterized by enhancing intestinal or brain bloodparacellular transport, the nanoparticle comprising a first component ofat least one bioactive agent, a second component of low molecular weightchitosan, and a third component that is negatively charged, wherein astabilizer is added to complex the at least one bioactive agent to thenegatively charged third component. In one embodiment, the stabilizer iszinc or calcium.

Monoclonal Antibody

Substances foreign to the body, such as disease-causing bacteria andviruses and other infectious agents, known as antigens, are recognizedby the body's immune system as invaders. Our natural defenses againstthese infectious agents are antibodies, proteins that seek out theantigens and help destroy them. Immunoglobulins are glycoproteins in theimmunoglobulin superfamily that function as antibodies. The termsantibody and immunoglobulin are often used interchangeably. They arefound in the blood and tissue fluids, as well as many secretions. Instructure, they are globulins (in the γ-region of proteinelectrophoresis). They are synthesized and secreted by plasma cells thatare derived from the B cells of the immune system. B cells are activatedupon binding to their specific antigen and differentiate into plasmacells. In some cases, the interaction of the B cell with a T helper cellis also necessary. The nominal size of an antibody is about 10 nm.

Humans have the ability to make antibodies able to recognize (by bindingto) virtually any antigenic determinant (i.e., epitope) and todiscriminate between even similar epitopes. An epitope is a small pieceof the antigen to which the antibody binds. Not only does this providethe basis for protection against disease organisms, but also it makesantibodies attractive candidates to target other types of moleculesfound in the body, such as receptors or other proteins present on thesurface of normal cells and molecules present uniquely on the surface ofcancer cells. Thus the remarkable specificity of antibodies makes thempromising agents for human therapy. It has been suggested to make anantibody that will bind only to the cancer cells in a patient, couple acytotoxic agent (e.g. a strong radioactive isotope or cancer-killingpaclitaxel) to that antibody, and then give the complex to the patientso it can seek out and destroy the cancer cells (and no normal cells).

Monoclonal antibodies are widely used as diagnostic and researchreagents. Their introduction into human therapy has been much slower. Insome in vivo applications, the antibody itself is sufficient. Once boundto its target, it triggers the normal effector mechanisms of the body,such as alerting other cells in the immune system to the presence of thecancer cells. In other cases, the monoclonal antibody is coupled toanother molecule, for example a fluorescent molecule to aid in imagingthe target or a strongly-radioactive atom, such as Iodine-131 to aid inkilling the target.

Some monoclonal antibodies that have been introduced into human medicineinclude those that suppress the immune system, such as:

-   -   Muromonab-CD3 (OKT3) and two humanized anti-CD3 monoclonals.        Bind to the CD3 molecule on the surface of T cells. Used to        prevent acute rejection of organ, e.g., kidney transplants. The        humanized versions show promise in inhibiting the autoimmune        destruction of beta cells in Type 1 diabetes mellitus.    -   Infliximab (Remicade®). Binds to tumor necrosis factor-alpha        (TNF-α). Shows promise against some inflammatory diseases such        as rheumatoid arthritis.    -   Omalizumab (Xolair®). Binds to IgE thus preventing IgE from        binding to mast cells. Shows promise against allergic asthma.    -   Daclizumab (Zenapax®). Binds to part of the IL-2 receptor        produced at the surface of activated T cells. Used to prevent        acute rejection of transplanted kidneys. Has also showed promise        against T-cell lymphoma.

Some monoclonal antibodies that have been introduced into human medicineinclude those that kill or inhibit malignant cells, such as:

-   -   Rituximab (Rituxan®). Binds to the CD20 molecule found on most        B-cells and is used to treat B-cell lymphomas.    -   Zevalin®. This is a monoclonal antibody against the CD20        molecule on B cells (and lymphomas) conjugated to either the        radioactive isotope indium-111 (¹¹¹In) or the radioactive        isotope yttrium-90 (⁹⁰Y)    -   Bexxar® (tositumomab). This is a conjugate of a monoclonal        antibody against CD20 and the radioactive isotope iodine-131        (¹³¹I). It is designed as a treatment for lymphoma.    -   Herceptin® (trastuzumab). Binds HER2, a receptor for epidermal        growth factor (EGF) that is found on some tumor cells (some        breast cancers, lymphomas). The only monoclonal so far that        seems to be effective against solid tumors.    -   Erbitux® (cetuximab). Blocks HER1, another epidermal growth        factor (EGF) receptor.    -   Mylotarg®. A conjugate of a monoclonal antibody that binds CD33,        a cell-surface molecule expressed by the cancerous cells in        acute myelogenous leukemia (AML) but not found on the normal        stem cells needed to repopulate the bone marrow. calicheamicin,        a complex oligosaccharide that makes double-stranded breaks in        DNA. Mylotarg® is the first immunotoxin that shows promise in        the fight against cancer.    -   LymphoCide. Binds to CD22, a molecule found on some B-cell        leukemias.    -   Alemtuzumab (MabCampath®). Binds to CD52, a molecule found on        white blood cells. Has produced complete remission of chronic        lymphocytic leukemia.    -   Lym-1 (Oncolym®). Binds to the HLA-DR-encoded histocompatibility        antigen that can be expressed at high levels on lymphoma cells.

Some monoclonal antibodies that have been introduced into human medicineinclude those that inhibit angiogenesis or perform other function, suchas:

-   -   Vitaxin. Binds to a vascular integrin (alpha-v/beta-3) found on        the blood vessels of tumors but not on the blood vessels        supplying normal tissues. In Phase II clinical trials, Vitaxin        has shown some promise in shrinking solid tumors without harmful        side effects.    -   Bevacizumab (Avastin®). Binds to vascular endothelial growth        factor (VEGF) preventing it from binding to its receptor.        Approved by the US FDA in February 2004 for the treatment of        colorectal cancers.    -   Abciximab (ReoPro®). Inhibits the clumping of platelets by        binding the receptors on their surface that normally are linked        by fibrinogen. Helpful in preventing reclogging of the coronary        arteries in patients who have undergone angioplasty.

In additional to the above monoclonal antibodies, other approvedmonoclonal antibodies may include, but not limited to, Basiliximab,Gemtuzumab ozogamicin, Ibritumomab, Palivizumab, Eculizumab, Adalimumab(Humira®), and Efalizumab.

By way of illustration, Natalizumab is a monoclonal antibodybioengineered from part of a mouse antibody to closely resemble a humanantibody. It is being marketed under the trade name Tysabri®. Theproduct is given intravenously once a month in a physician's office fortreating multiple sclerosis (MS) to reduce the frequency of symptomflare-ups or exacerbations of the disease. MS is a chronic, oftendisabling disease of the brain and spinal cord.

By way of illustration, Rituximab targets a protein called CD20, foundon the surface of type B lymphocytes. These are one of the main types ofwhite blood cells, and the cell at fault in most cases of non-Hodgkin'slymphoma. Although the drug attacks both cancerous and non-canceroustype B cells, the body quickly replaces them with healthy lymphocytes.Monoclonal antibodies have been designed to recognize certain proteinsfound on the surface of some cancer cells. Once the monoclonal antibodyhas recognized the protein, it locks on to it and triggers the body'simmune system to attack the cancer cells, without affecting other cellsin the body. Sometimes it instructs the cells to destroy themselves.

One possible treatment for cancer involves monoclonal antibodies (mAb)that bind only to cancer cell-specific antigens and induce animmunological response against the target cancer cell. Such mAb couldalso be modified for delivery of a toxin, radioisotope, cytokine orother active conjugate; it is also possible to design bispecificantibodies that can bind with their Fab regions both to target antigenand to a conjugate or effector cell. In fact, every intact antibody canbind to cell receptors or other proteins with its Fc region.

Antibody Structure and Binding with Antigen

Immunoglobulins are heavy plasma proteins, often with added sugar chainson N-terminal (all antibodies) and occasionally O-terminal (IgA1 andIgD) amino acid residues. According to differences in their heavy chainconstant domains, immunoglobulins are grouped into five classes, orisotypes: IgG, IgA, IgM, IgD, and IgE. Other immune cells collaboratewith antibodies to eliminate pathogens depending on which IgG, IgA, IgM,IgD, and IgE constant binding domain receptors it can express on itssurface.

The basic unit of each antibody is a monomer. An antibody can bemonomeric, dimeric, trimeric, tetrameric, pentameric, etc. The monomeris a “Y”-shape molecule that consists of two identical heavy chains andtwo identical light chains connected by disulfide bonds. There are fivetypes of heavy chain: γ, δ, α, μ and ε. They define classes ofimmunoglobulins. Each heavy chain has a constant region, which is thesame by all immunoglobulins of the same class, and a variable region,which differs between immunoglobulins of different B cells, but is thesame for all immunoglobulins produced by the same B cell. Heavy chainsγ, α and δ have the constant region composed of three domains but have ahinge region; the constant region of heavy chains μ and ε is composed offour domains. The variable domain of any heavy chain is composed of onedomain. These domains are about 110 amino acids long. There are alsosome amino acids between constant domains.

The “Y”-shaped monomer has two heavy and two light chains. Together thisgives six to eight constant domains and four variable domains. Each halfof the forked end of the “Y” is called an Fab fragment. It is composedof one constant and one variable domain of each the heavy and the lightchain, which together shape the antigen binding site at the aminoterminal end of the monomer. The two variable domains bind theirspecific antigens. The enzyme papain cleaves a monomer into two Fab(fragment antigen binding) fragments and an Fc (fragment crystallizable)fragment. The enzyme pepsin cleaves below hinge region, so a Fabfragment and a Fc fragment is formed. Together, the antibodies in anorganism can bind a wide variety of foreign antigens.

The antibodies have two primary functions: they bind antigens and theycombine with different immunoglobulin receptors specific for them andexert effector functions. These receptors are isotype-specific, whichgives a great flexibility to the immune system, because differentsituations require only certain immune mechanisms to respond toantigens. By “Affinity” is herein defined as the binding strength of theantibody to the antigen. By “Avidity” is herein defined as the number ofantigen binding sites.

Antibodies exist freely in the bloodstream or bound to cell membranes.They are part of the humoral immune system. Antibodies exist in clonallines that are specific to only one antigen, e.g., a virus hull protein.In binding to such antigens, they can cause agglutination andprecipitation of antibody-antigen products primed for phagocytosis bymacrophages and other cells, block viral receptors, and stimulate otherimmune responses, such as the complement pathway. Antibodies thatrecognize viruses can block these directly by their sheer size. Thevirus will be unable to dock to a cell and infect it, hindered by theantibody. They can also agglutinate them so the phagocytes can capturethem. Antibodies that recognize bacteria mark them for ingestion byphagocytes, a process called opsonization. Together with the plasmacomponent complement, antibodies can kill bacteria directly. Theyneutralize toxins by binding with them.

In biochemistry, antibodies are used for immunological identification ofproteins, using the Western blot method. A similar technique is used inELISPOT and ELISA assays, in which detection antibodies are used todetect cell secretions such as cytokines or antibodies. Antibodies arealso used to separate proteins (and anything bound to them) from theother molecules in a cell lysate. A Western blot (a.k.a immunoblot) is amethod in molecular biology/biochemistry/immunogenetics to detectprotein in a given sample of tissue homogenate or extract. It uses gelelectrophoresis to separate denatured proteins by mass. The proteins arethen transferred out of the gel and onto a membrane (typicallynitrocellulose), where they are “probed” using antibodies specific tothe protein. As a result, researchers can examine the amount of proteinin a given sample and compare levels between several groups.

The Enzyme-Linked ImmunoSorbent Assay (ELISA for short) is a biochemicaltechnique used mainly in immunology to detect the presence of anantibody or an antigen in a sample. It utilizes two antibodies, one ofwhich is specific to the antigen and the other of which is coupled to anenzyme. This second antibody gives the assay its “enzyme-linked” name,and will cause a chromogenic or fluorogenic substrate to produce asignal. Because the ELISA can be performed to evaluate either thepresence of antigen or the presence of antibody in a sample, it is auseful tool both for determining serum antibody concentrations (such aswith the Human Immunodeficiency Virus, HIV test or West Nile Virus) andalso for detecting the presence of antigen. ELISPOT is an immunologicalassay based on ELISA. Basically, the difference between the two is thatin ELISA, the substance containing the “unknown” is stuck at the bottomof the well, whereas in ELISPOT the substance with the “unknown” isplaced in the well after the bottom of the well has been coated withcytokine-specific antibody. ELISPOT is a method for detecting cytokineproduction at the single cell level whereas ELISA is a sensitive andspecific method for quantification of different cytokines.

Antibody detection kits, such as the ExtrAvidin® Staining Kits (Sigma,St Louis, Mo.) are used to assay the presence of monoclonal antibody innanoparticles. These kits comprise universal reagents for use withprimary antibodies in immunohistology, ELISA, and immunoblotting.ExtrAvidin is a unique form of avidin that combines the high specificityand affinity of avidin for biotin with low non-specific binding atphysiological pH. ExtrAvidin alkaline phosphatase and peroxidase enzymesthus exhibit high sensitivity with low background. For example,monoclonal anti-goat IgG antibodies in EXTRA-1 show no cross-reactivitywith human IgG. Further, affinity isolated antibodies in EXTRA-2A andEXTRA-3A etc. have been adsorbed with human IgG and IgM to minimizecross-reactivity. Biotinylated antibodies contain a spacer whichimproves accessibility for the ExtrAvidin conjugates.

EXAMPLE NO. 18

Nanoparticles with Monoclonal Antibody

One aspect describes nanoparticles for oral administration in a patientcomprising a positively charged shell substrate, a negatively chargedcore substrate, and a bioactive agent encapsulated within thenanoparticles, wherein the bioactive agent is monoclonal antibody. Inone embodiment, the bioactive agent is mixed with, conjugated to, orcoupled to the core substrate. In another embodiment, the bioactiveagent is totally encapsulated within the nanoparticles.

Glycosaminoglycan (GAG), heparin, α-PGA, or γ-PGA is a negativelycharged substrate that serves to bind with positively charged chitosansubstrate to form a nanoparticle. Some aspects of the invention relateto GAG containing nanoparticles for oral administration. In oneembodiment, GAG serves as at least a portion of the core substrate withchitosan serves as at least a portion of the shell substrate, whereinGAG conjugates at least one bioactive agent as disclosed herein, forexample, monoclonal antibodies. In preparation, nanoparticles wereobtained upon addition of aqueous solution (2 ml) of GAG and monoclonalantibody “Bevacizumab”, using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a low-MW CS aqueous solution(pH 6.0, 10 ml) with excess CS concentrations under magnetic stirring atroom temperature.

Nanoparticles were collected by ultracentrifugation at 38,000 rpm for 1hour and coded mAb-NP, which is formed of shell substrate chitosan, coresubstrate GAG with encapsulated monoclonal antibody. The monoclonalantibody is wholly or substantially totally encapsulated in thenanoparticles. The nanoparticles have an average diameter from about 50nm to about 500 nm. In a preferred embodiment, the nanoparticles arenanoshells having biodegradable chitosan as a shell substrate. Thenanoparticles may further comprise a core substrate that mixes with themonoclonal antibody or monoclonal antibody anti-cancer drug. In clinicalsettings, the method of treating a tumor with a monoclonal antibodyanti-cancer drug that is released from the nanoparticles comprises astep of binding the anti-cancer drug to a cell or tissue of the patient,wherein the binding is by the formation of an antigen-antibody complexor the formation of a ligand-receptor complex. In one embodiment, thecell or tissue is cancerous. As described above, the nanoparticlescomprise a positively charged shell substrate and a negatively chargedcore substrate, wherein the nanoparticles have a surface charge fromabout +15 mV to about +50 mV.

Avastin is a recombinant humanized monoclonal IgG1 antibody that bindsto and inhibits the biologic activity of human vascular endothelialgrowth factor (VEGF) in in vitro and in vivo assay systems, and is alsosometimes described as a targeted therapy. Avastin is a particular typeof targeted therapy called anti-angiogenic therapy that may interferewith the growth of new blood vessels, which provide nutrients to thetumor. Avastin is the first anti-angiogenic therapy in combination withfirst-line chemotherapy proven to help many people with metastaticcolorectal cancer live longer. In clinical trials, patients takingAvastin in combination with chemotherapy experienced many benefitscompared with those taking chemotherapy alone. In order to grow andspread, tumors need a constant supply of oxygen and other nutrients.Tumors get this supply by creating their own network of blood vessels.This process is called angiogenesis.

To start angiogenesis, a tumor sends out signals to nearby bloodvessels. These signals cause new blood vessels to grow toward the tumor.Once these new vessels reach the tumor, they provide the supply of bloodthat provides oxygen and other nutrients to the tumor. Avastin isthought to work by interfering with the signals that cause angiogenesis.Avastin is given by infusion. Bevacizumab is produced in a ChineseHamster Ovary mammalian cell expression system in a nutrient mediumcontaining the antibiotic gentamicin and has a molecular weight ofapproximately 149 kilodaltons. AVASTIN is a clear to slightlyopalescent, colorless to pale brown, sterile, pH 6.2 solution forintravenous (IV) infusion.

The obtained mAb-NP in Example no. 18 is broken up by extremestirring/beating and the supernatant is assayed by ELISA to confirm thepresence of Bevacizumab antibody in the sample with endothelial growthfactor as target binding antigen.

Some aspects of the invention relate to a method of delivering amonoclonal antibody protein to a target tissue or a tumor of a patient,comprising the steps of: providing the monoclonal antibody protein tothe target tissue or the tumor, wherein the monoclonal antibody proteinis encapsulated within nanoparticles; administering the nanoparticles tothe patient orally; and treating the target tissue or the tumor with themonoclonal antibody protein that is released from the nanoparticles. Onepreferred aspect of the invention relate to a method of treating atarget tissue or organ of a patient with a monoclonal antibody,comprising the steps of: providing the monoclonal antibody to the tissueor organ, wherein the monoclonal antibody is encapsulated withinnanoparticles; administering the nanoparticles to the patient orally;and treating the target tissue or organ with the monoclonal antibodythat is sustained released from the nanoparticles. In one embodiment,the monoclonal antibody protein is an anti-cancer drug for the tumor. Inanother embodiment, the monoclonal antibody is Adalimumab for treatingrheumatoid arthritis or psoriatic arthritis. In still anotherembodiment, the monoclonal antibody protein is Bevacizumab for treatingtumor or cancer. FIG. 19 shows a schematic composition of a nanoparticlewith a shell substrate and a core substrate having a monoclonalantibody. The method may further comprise a step of delivering thenanoparticles to the target tissue or tumor through intestinalabsorption. In a preferred embodiment, the anti-cancer monoclonalantibody is directed against tumor vasculature. Some aspect providesthat the target tumor is in an organ selected from the group consistingof breast, lung, brain, liver, skin, kidney, GI organ, prostate, bladderand gynecological organ.

EXAMPLE NO. 19

Nanoparticles with Monoclonal Antibody Adalimumab

Humira® (Adalimumab) is a recombinant human IgG1 monoclonal antibodyspecific for human tumor necrosis factor (TNF). It consists of 1330amino acids and has a molecular weight of approximately 148 kilodaltons.The solution of Humira is clear and colorless, with a pH of about 5.2and contains 40 mg Adalimumab in each 0.8 mL of single-use syringe. TNFis a naturally occurring cytokine that is involved in normalinflammatory and immune responses. Elevated levels of TNF are found inthe synovial fluid of rheumatoid arthritis and psoriatic arthritispatients and play an important role in both the pathologic inflammationand the joint destruction that are hallmarks of these diseases.

Clinically, after treatment with Humira, a rapid decrease in levels ofacute phase reactants of inflammation (C-reactive protein, erythrocytesedimentation rate, and serum cytokines IL-6) was observed compared tobaseline in patients with rheumatoid arthritis. Serum levels of matrixmetalloproteinases (MMP-1 and MMP-3) that produce tissue remodelingresponsible for cartilage destruction were also decreased after Humiraadministration. Humira is administered subcutaneously and/orintravenously. Adalimumab may also be effective against plaquepsoriasis, ankylosing spondylitis or Crohn's disease.

In preparation of nanoparticles encapsulating Adalimumab, the processcomprises addition of aqueous solution (2 ml) of GAG and monoclonalantibody “Adalimumab” by using a pipette (0.5-5 ml, PLASTIBRAND®,BrandTech Scientific Inc., Germany), into a chitosan aqueous solution(pH 6.0, 10 ml) with excess CS concentrations under magnetic stirring atroom temperature. Nanoparticles were collected by ultracentrifugation at38,000 rpm for 1 hour and were assayed by ELISA technique to confirm thepresence of Adalimumab monoclonal antibody in the nanoparticles withtarget binding antigen. One aspect of the invention describes a methodof delivering a monoclonal antibody to a target tissue or organ of apatient, comprising the steps of: providing the monoclonal antibody tothe tissue or organ, wherein the monoclonal antibody is encapsulatedwithin nanoparticles; administering the nanoparticles to the patientorally; and treating the target tissue or organ with the monoclonalantibody that is controlled released from the nanoparticles. The methodfurther comprises a step of delivering the nanoparticles to the targettissue or organ through intestinal absorption.

The following ELISA method has been adopted for monoclonal antibodyassay. The stock solutions include: PBS (20 mM NaPi pH=7.5, 150 mMNaCl), PT (0.1% Tween 20 in PBS), Blocking solution (3% BSA in PT; storeat −20° C.), and Developing solution (10 mg o-phenylenediamine in 25 mlof a buffer with pH=5.5 plus 12 μl of 30% H₂O₂. This solution is lightsensitive and is prepared just before use).

The ELISA procedures include:

-   -   1. Coat the plate wells with 50 μl of antigen (0.2 μg/ml in        PBS). Incubate one hour at room temperature;    -   2. Wash the plate (discard the well solution, add 200 μl of PT        per well and wait about 2 min. Repeat this cycle another 2 times        and discard the late well solution);    -   3. Block the plate wells with 150 μl of blocking solution.        Incubate one hour at room temperature;    -   4. Discard the blocking solution and add 50 μl of the hybridoma        supernatants in different wells. Incubate 30 min at room        temperature;    -   5. Wash the plate;    -   6. Add 50 μl of the peroxidase-coupled antimouselgs antibody        (1/5000 in blocking solution). Incubate 30 min at room        temperature;    -   7. Wash the plate;    -   8. Incubate the plate wells with 100 μl of developing solution        until color change is evident;    -   9. Stop the reaction by adding 50 μl of 2.5M H₂SO₄ per well; and    -   10. Measure the absorbance at 492 nm.

Adalimumab is an injectable protein that blocks the inflammatory effectsof tumor necrosis factor alpha (TNF alpha) in rheumatoid arthritis. Twoother injectable drugs—Infliximab (Remicade®) and etanercept(Enbrel®)—also block TNF alpha. One aspect of the invention provides ananoparticle comprising a shell substrate of chitosan, a core substrate,and an encapsulated bioactive agent that blocks TNF-alpha, wherein thebioactive agent is selected from the group consisting of Adalimumab,Infliximab, etanercept, and the like. Adalimumab was constructed from afully human monoclonal antibody, while infliximab is a mouse-humanchimeric antibody and etanercept is a TNF receptor-IgG fusion protein.Inflammation is the body's reaction to injury and is a necessary processfor the repair of injury. TNF is a protein that the body produces whenthere is inflammation. The TNF promotes inflammation and the signs ofinflammation, which, in the case of arthritis, include fever as well aspain, tenderness, and swelling of joints. The unchecked inflammation ofrheumatoid arthritis eventually leads to destruction of the joints.Adalimumab is a synthetic (man-made) protein, similar to human protein,that binds to TNF in the body and thereby blocks the effects of TNF. Asa result, inflammation and fever as well as the pain, tenderness andswelling of joints are reduced in patients with rheumatoid arthritis. Inaddition, the progressive destruction of the joints is slowed orprevented. Adalimumab was approved by the FDA in December 2002.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims. Many modifications and variations are possible inlight of the above disclosure.

1. A pharmaceutical composition of nanoparticles for oral administrationin a patient, said nanoparticles comprising a shell portion ofbiodegradable chitosan that is positively charged, a core portion ofnegatively charged substrate of polyglutamic acid (PGA) that isneutralized with a portion of positively charged chitosan, and amonoclonal antibody loaded within said nanoparticles, wherein saidnanoparticles further comprise at least one paracellular transportenhancer.
 2. The pharmaceutical composition of claim 1, wherein said PGAis γ-PGA.
 3. The pharmaceutical composition of claim 1, wherein asurface of said nanoparticles is characterized with a positive surfacecharge.
 4. The pharmaceutical composition of claim 1, wherein saidnanoparticles have an average diameter from about 50 nm to about 500 nm.5. The pharmaceutical composition of claim 1, wherein said nanoparticleshave a surface charge from about +15 mV to about +50 mV.
 6. Thepharmaceutical composition of claim 1, wherein said nanoparticles areencapsulated in a capsule.
 7. The pharmaceutical composition of claim 6,wherein said capsule is treated with an enteric coating.
 8. Thepharmaceutical composition of claim 1, wherein said monoclonal antibodyis conjugated to said negatively charged substrate.
 9. Thepharmaceutical composition of claim 1, wherein said monoclonal antibodybinds only to cancer cells in the patient.
 10. The pharmaceuticalcomposition of claim 1, wherein the monoclonal antibody suppress theimmune system of the patient.
 11. The pharmaceutical composition ofclaim 1, wherein the monoclonal antibody kills or inhibits malignantcells in the patient.
 12. The pharmaceutical composition of claim 1,wherein said nanoparticles are formed via a simple and mildionic-gelation method.
 13. The pharmaceutical composition of claim 1,wherein said paracellular transport enhancer is a Ca²⁺ chelator.